ES2668222T3 - Herbicide tolerant plants of HPPD - Google Patents

Abstract

A chimeric gene comprising a coding sequence operatively linked to a plant-expressible promoter, the latter being a heterologous regulatory element to the coding sequence, characterized in that the coding sequence comprises a nucleic acid sequence encoding an HPPD protein comprising the sequence of HPPD amino acids of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387 or a protein with a sequence identity of at least 80% with the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, which shows the properties of catalyzing the conversion of para-hydroxyphenylpyruvate to homogentisate and being less sensitive to an herbicide inhibitor of HPPD than the endogenous HPPD of the host plant.

Description

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DESCRIPTION

Plants that tolerate herbicide inhibitors of HPPD Introduction

The present invention relates to nucleic acid sequences encoding a hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, abbreviated herein HPPD) obtained from bacteria belonging to the genus Kordia, as well as a chimeric gene comprising said acid sequence nucleic acids and the use of said nucleic acid sequences, proteins or chimeric genes to obtain plants that are tolerant to HPPD inhibitory herbicides.

Background

HPPDs are enzymes that catalyze the reaction in which a para-hydroxyphenylpyruvate (abbreviated herein as HPP), a degradation product of tyrosine, is transformed into a homogentisate (abbreviated herein as HG), the precursor in tocopherol and plastoquinone plants (Crouch NP et al. (1997) Tetrahedron, 53, 20, 6993-7010, Fritze et al. (2004), Plant Physiology 134: 1388-1400). Tocopherol acts as an antioxidant associated with the membrane. Plastoquinone first acts as an electron transporter between the PSII and the cytochrome b6 / f complex and secondly it is a redox cofactor for phytoene desaturase, which is involved in the biosynthesis of carotenoids.

So far, more than 700 nucleic acid sequences from various organisms present in the NCBI database were recorded as encoding a putative protein that had an HPPD domain and included the sequence described under accession number A9DQF2 which it is given in the UniProtKB / TrEMBL database as well as under the accession number ZP02161490 that is given in the NCBI protein database. However, for most of these, including the sequence corresponding to access number A9DQF2 /

ZP02161490, it has not been demonstrated that this protein could have an enzymatic activity of HPPD either in an in vitro assay or in an in-plant approach, nor that said HPPD protein can confer to herbicide inhibitor HPPD herbicides, when expressed in a plant. Several HPPD proteins and their primary sequences have been described in the state of the art, in particular the HPPD proteins of bacteria such as Pseudomonas (Rüetschi et al., Eur. J. Biochem., 205, 459-466, 1992, document

WO 96/38567), of plants such as Arabidopsis (WO 96/38567, Genebank AF047834), carrots (Wo 96/38567, Genebank 87257), Avena sativa (WO 02/046387), wheat (WO 02 / 046387), Brachiaria platyphylla (document WO 02/046387), Cenchrus echinatus (document WO 02/046387), Lolium rigidum (document WO 02/046387), Festuca arundinacea (document wO 02/046387), Setaria faberi (document WO 02 / 046387), Eleusine indica (WO 02/046387), sorghum (Wo 02/046387), Coccicoides (Genebank COITRP), of Coptis japonica (WO 06/132270), Chlamydomonas reinhardtii (Spanish patent document ES 2275365), or of mammals such as a mouse or a pig.

Most of the plants synthesize tyrosine through arrogenate (Abou-Zeid et al. (1995), Applied Env Microb 41: 1298-1302; Bonner et al. (1995), Plant Cell Physiol. 36, 1013-1022 ; Byng et al. (1981), Phytochemistry 6: 12891292; Connely and Conn (1986), Z. Naturforsch 41c: 69-78; Gaines et al. (1982), Plant 156: 233-240). In these plants, HPP derives only from the degradation of tyrosine. On the other hand, in organisms such as Saccharomyces cerevisiae yeast or Escherichia coli bacteria, HPP is a precursor to tyrosine and is synthesized by the action of an enzyme, prefenate dehydrogenase (hereinafter referred to as PDH) , which converts prefenate to HPP (Lingens et al., (1967) European J. Biochem 1: 363-374; Sampathkumar and Morrisson (1982), Bioch Biophys Acta 701: 204-211). In these organisms, the production of HPP is therefore directly connected with the aromatic amino acid biosynthesis pathway (shikimato route) and not with the tyrosine degradation pathway.

The inhibition of an HPPD leads to a decoupling of photosynthesis, a deficiency in terms of accessory pigments that harvest light and, most importantly, the destruction of chlorophyll by UV radiation and reactive oxygen species (bleaching) due to lack of protection against light that is normally provided by carotenoids (Norris et al. (1995), Plant Cell 7: 2139-2149). Bleaching of photosynthetically active tissues leads to growth inhibition and death of plants.

Some molecules that inhibit HPPD, and that specifically bind to the enzyme in order to inhibit the transformation of HPP into homogentisate, have proven to be very effective selective herbicides.

At present, most of the HPPD inhibitor herbicides that are available belong to one of these four chemical families:

1) tricetonas, for example sulcotrione [ie 2- [2-chloro-4- (methylsulfonyl) benzoyl] -1,3-cyclohexanedione], mesotrione [ie 2- [4- (methylsulfonyl) -2-nitrobenzoyl] -1,3-cyclohexanedione]; trembling [ie 2- [2-chloro-4-

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(methylsulfonyl) -3 - [(2,2,2-trifluoroethoxy) methyl] benzoyl] -1,3-cidohexanedione]; tefuryltrione [ie 2- [2-chloro-4- (methylsulfonyl) -3 - [[(tetrahydro-2-furanyl) methoxy] methyl] benzoyl] -1,3-cidohexanedione]]; bicyclopyrone [ie 4- hydroxy-3 - [[2 - [(2-methoxyethoxy) methyl] -6- (trifluoromethyl) -3-pyridinyl] -carbonyl] bicido [3.2.1] oct-3-en-2- ona]; benzobicyclone [ie 3- (2-chloro-4-mesylbenzoyl) -2-phenylthiobicyclo [3.2.1] oct-2-en-4-one]

2) the dikettonitriles, for example 2-cyano-3-cyclopropyl-1- (2-methylsulfonyl-4-trifluoromethylphenyl) -propane-1,3-dione and 2- cyano-1- [4- (methylsulfonyl) -2- trifluoromethylphenyl] -3- (1-methylcyclopropyl) propane-1,3-dione;

3) isoxazoles, for example isoxaflutol [ie (5-cyclopropyl-4-isoxazolylH2- (methylsulfonyl) -4- (trifluoromethyl) phenyl] methanone]. In plants, isoxaflutol is rapidly metabolized into DKN, which is a compound dicetonitrile that exhibits the inhibitory property of HPPDs; and

4) pyrazolinates, for example topramezone [ie [3- (4,5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl] (5- hydroxy-1-methyl-1H-pyrazole- 4-yl) methanone], and pyrasulfotol [(5-hydroxy-1,3-dimethylpyrazol-4-yl- (2-mesyl-4- trifluoromethylphenyl) -methanone]; pyrazophen [2- [4- (2,4- dichlorobenzoyl) -1,3-dimethylpyrazol-5-yloxy] acetophenone].

These HPPD inhibitor herbicides can be used against herbaceous and / or broadleaf weeds in the presence of cultivated plants, which have a metabolic tolerance, such as corn (Zea mays), in which they are rapidly degraded (Schulz and col., (1993) FEBS letters, 318, 162-166; Mitchell et al., (2001) Pest Management Science, Vol 57, 120-128; Garcia et al., (2000) Biochem., 39, 7501- 7507; Pallett et al., (2001) Pest Management Science, Vol 57, 133-142). In order to expand the reach of these HPDD inhibitor herbicides, several efforts have been developed to confer to plants, particularly to plants without metabolic tolerance or with a metabolic tolerance of low yield, a tolerance level that is acceptable in agronomic conditions in the field.

Together with the attempt to avoid the production of homogentisate mediated by an HPPD (US 6,812,010), overexpression of the sensitive enzyme has been performed, so that sufficient amounts of the target enzyme are produced in the plant that are sufficient in relation to the herbicide (document WO96 / 38567). Overexpression of an HPPD resulted in a better tolerance before the outbreak for the diketonitrile derivative (DKN) of isoxaflutol (IFT), but the tolerance was not sufficient to have a tolerance to an after-out treatment (Matringe et al., (2005), Pest Management Science 61: 269-276).

A third strategy was to mutate the HPPD in order to obtain a target enzyme that, while retaining its properties of catalyzing the transformation of HPP into homogentisate, is less sensitive to HPPD inhibitors than is native HPPD before the mutation

This strategy has been successfully applied for the production of 2-cyano-3-cyclopropyl-1- (2- methylsulfonyl-4-trifluoromethylphenyl) -propane-1,3-dione and 2-cyano-1- tolerant plants [4- (methylsulfonyl) -2-trifluoromethylphenyl] -3- (1- methylcyclopropyl) propane-1,3-dione (EP496630), which are two HPPD inhibitory herbicides belonging to the dikettonitrile family (wO document 99/24585). The Pro215Leu, Gly336Glu, Gly336Ile and more particularly Gly336Trp (the mutated amino acid positions are indicated with reference to Pseudomonas HPPD) were identified as mutations that are responsible for an increased tolerance to a treatment before the outbreak with these dikettonitrile herbicides, without causing an alteration of enzyme activity.

More recently, it has been shown that the introduction of a Pseudomonas HPPD gene into the genome of tobacco and soy plastids is more effective than a nuclear transformation, conferring even tolerance to the application of isoxaflutol after the outbreak (Dufourmantel et al., 2007, Plant Biotechnol J.5 (1): 118-33).

In WO 04/024928, the authors of the invention have sought to increase the biosynthesis of prenylquinone (for example, synthesis of plastoquinones and tocopherols) in plant cells by increasing the flow of the HPP precursor within the cells of these plants. . This has been done by connecting the synthesis of said precursor with the "shikimato" route by overexpression of a PDH enzyme. They have also observed that the transformation of plants with a gene encoding a PDH enzyme makes it possible to increase the tolerance of said plants to HPPD inhibitors.

In patent application WO 2009/144079, a nucleic acid sequence encoding a hydroxyphenylpyruvate dioxygenase (HPPD) mutated at position 336 of the HPPD protein of Pseudomonas fluorescens and its use to obtain plants that are tolerant to herbicide inhibitors are described of the HPPD.

In WO 2002/046387, several domains of HPPD proteins that originate from plants that may be relevant to confer tolerance to various HPPD inhibitor herbicides have been identified but no in-plant or biochemical data has been shown to confirm the impact of the functions of the domains just described.

In WO 2008/150473, the combination of two different tolerance mechanisms was given as an example - a modified Avena sativa gene encoding a mutant HPPD enzyme and a CYP450 of corn monooxygenase (nsf1 gene) in order to obtain an improved tolerance to herbicides inhibitors of HPPD, but no data have been described that demonstrate the synergistic effects based on the combination of both proteins.

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Despite these successes for the development of plants that show tolerance for various HPPD inhibitor herbicides described above, it is still necessary to develop and / or improve plant tolerance to newer HPPD inhibitor agents or several different ones, particularly those that inhibit HPPD that belong to the classes of tricetonas (for example sulcotrione, mesotrione, tembotriona, benzobiciclone and bicyclopyrone) and pyrazolinates (for example, topramezone and pyrasulfotol).

Description

The present invention therefore relates to the generation of transgenic plants that contain a gene encoding an HPPD protein obtainable or obtained from an organism belonging to the Kordia genus, and variants or mutants thereof, more especially to a gene from an organism that belongs to the species Kordia algicide and variants or mutants thereof, which encodes an HPPD enzyme that shows the properties of catalyzing the conversion of a para-hydroxyphenylpyruvate into a homogentisate and whose plants are less sensitive to agents that inhibit the HPPD that some plants that contain said transgene that encodes an HPPD. Kordia genes encoding HPPD proteins were selected as excellent tolerant candidates for HPPD inhibiting agents due to their high divergences in amino acid composition at relevant positions for tolerance to HPPD inhibiting agents, as It determines experimentally and structurally the HPPD protein compared to the sensitive Arabidopsis HPPD protein that was taken as the sensitive reference molecule to HPPD inhibiting agents.

In one embodiment, the present invention relates to an HPPD protein referred to herein as "the HPPD protein of the present invention" or "the Kordia HPPD protein", which is an HPPD protein with an identity of its amino acid sequence of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%; at least 97%; at least 98%, or at least 99% with respect to the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, particularly with respect to the amino acid sequence of any one of SEQ ID NO. 4, 5, 6 or 7, preferably from SEQ ID NO. 6. The disclosure refers to an HPPD protein referred to herein as "the HPPD protein of the present invention" or "the Kordia HPPD protein", which is an HPPD protein with an identity of its amino acid sequence. at least 75%, at least 80%, at least 85%, at least 90%, at

minus 95%; at least 97%; at least 98%, or at least 99% with respect to the amino acid sequence of the

SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, particularly with respect to the amino acid sequence of any one of the sEq ID NO. 4, 5, 6, 7, preferably from SEQ ID NO. 6, and in that any of the amino acids from position 195 to position 387 of SEQ ID NO. 4 can be corrected for any amino acid present in nature, preferably it can be any conservative substitution. The disclosure further relates to an HPPD protein referred to herein as "the HPPD protein of the present invention" or "the Kordia HPPD protein", which is an HPPD protein with an identity of its amino acid sequence of at least 75%, at least 80%, at least 85%, at least 90%, at

minus 95%; at least 97%; at least 98%, or at least 99% with respect to the amino acid sequence of the

SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, particularly with respect to the amino acid sequence of any one of the sEq ID NO. 4, 5, 6, 7, preferably from SEQ ID NO. 6, and having one or more of the following amino acids in the position defined by their number (which refers to the number of SEQ ID NO. 4) given in parentheses, that is His (193), Ser (236), Asn (251), Gln (275), His (276), Tyr (305), Gln (340), Phe (353), Glu (355), Gly (366), and Asn (369). The disclosure further relates to an HppD protein referred to herein as "the HPPD protein of the present invention" or "the Kordia HPPD protein", which is an HPPD protein with an identity of its amino acid sequence of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%; at least 97%; at least 98%, or at least 99% with respect to the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, particularly with respect to the amino acid sequence of any one of SEQ ID NO. 4, 5, 6, 7, preferably from SEQ ID NO. 6, and in the respective positions given in the second column of Table (i) the amino acids originally present may be substituted for any of the amino acids listed in column 3 of Table (i).

Amino acid in SEQ ID

NO.4

Val

Ile

Ile

Phe

Leu

Lys

Met

Table (i):

SEQ position

ID NO.4

195

219

220 222 234 237 239

Substitutions Thr, Cys, Ala, Gly

Phe, Tyr, Leu, Val, Ala, Gln, Glu, Asp, Gly, Thr, Ser, Met, Arg, Lys Ala, Trp, Leu, Ser, Arg, Lys, His, Asp, Glu, Pro, Gly, Asn Val, Ile, Ala, Leu, Trp, Met, Gln, His Met, Val

Ala, Val, Leu, Met, Ile, Arg, Gln, Tyr Leu, Val, Ile, Ala

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(continuation)

 Amino acid in SEQ ID NO.4

 Position in SEQ ID NO.4 Substitutions

 Be

 240 Ala, Thr, Val, Arg, Lys, Glu, Leu, Ile, Met, His

 To

 372 Glu, Gln, Ser, Val, Phe, Thr

 Leu

 373 Arg

The disclosure further relates to an HPPD protein referred to herein as "the HPPD protein of the present invention" or "the Kordia HPPD protein", which is an HPPD protein with an amino acid sequence identity of at minus 75%, at least 80%, at least 85%, at least 90%, at least 95%; at least 97%; at least 98%, or at least 99% with respect to the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to 387, particularly with respect to the amino acid sequence of any one of SEQ ID NO. 4, 5, 6, 7, preferably from SEQ ID NO. 6, and in the respective positions given in the second column of Table (ii) the amino acids originally present may be substituted for any of the amino acids listed in column 3 of Table (ii).

Table (ii):

Acid amino in SEQ ID

NO.4

Be

Val

Pro

Leu

Leu

Val

Gly

Position in SEQ ID NO.4 221 238 249 299 329

367

368

Substitutions

Glu, Thr, Tyr, Phe, His, Gln, Asn, Gly, Leu, Met, Val, Arg, Ile Ala, Thr

Ala, Val, Thr, Asn, Ile,

Met, Ile, Asn Met

Anyone except Pro Ala, Pro, Val, Thr, Met

The disclosure further relates to an HPPD protein referred to herein as "the HPPD protein of the present invention" or "the Kordia HPPD protein", which is an HPPD protein with an identity of its amino acid sequence of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%; at least 97%; at least 98%, or at least 99% with respect to the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to 387, particularly with respect to the amino acid sequence of any one of SEQ ID NO. 4, 5, 6, 7, preferably SEQ ID NO. 6, and in the respective positions given in the second column of Table (iii) the amino acids originally present may be substituted for any of the amino acids listed in column 3 of Table (iii).

Table (iii)

 Acid amino in SEQ ID NO.4

 Position in SEQ ID NO.4 Substitutions

 Be

 221 Glu, Thr, Arg, Tyr

 Val

 238 Wing

 Pro

 249 Ala, Val, Thr

 Leu

 299 Met

 Leu

 329 Met

 Val

 367 Ile, Ala, Val, Leu, Lys

 Gly

 368 Wing

The invention uses a protein with substituted, deleted or added amino acids compared to the sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, such as a fusion protein with a transit peptide, or a protein with amino acid changes in the sequence of SEQ ID NO. 4 that retains the enzymatic function of an HPPD protein, and that still confers a tolerance to HPPD when expressed in plants, preferably a tolerance to HPPD of a range comparable to that conferred by the protein of SEQ ID NO. 4. This includes variant or mutant proteins derived from the protein of SEQ ID NO. 4, such as any of the proteins of SEQ ID NO. 5, 6 or 7, particularly that mutant or variant that is less sensitive than the endogenous HPPD of the host plant to an inhibitor herbicide of the HPPD of the isoxazole class, dikettonitriles, tricetonas or pyrazolinates, preferably that mutant

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or variant that confers an agronomically relevant herbicide tolerance to a host plant that expresses it when an HPPD inhibitor herbicide of the isoxazole class, dikettonitriles, tricetonas and / or pyrazolinates, particularly any taken from mesotrione, tremotrione, isoxaflutol or bicyclopirone it is applied on said plants, more particularly when it is applied after the outbreak. This also includes a protein that comprises an active portion of the sequence of SEQ ID NO. 4, whose portion confers a tolerance to HPPD inhibiting agents when expressed in plants. This includes a protein with substantially the same amino acid sequence as the sequence of SEQ ID NO. 4, such as a protein with the amino acid sequence of any one of SEQ ID NO. 4 to 7. This includes isolated proteins as defined below, and also proteins, such as the protein of SEQ ID NO. 4, in which certain amino acids have been replaced by similar amino acids as defined below, preferably conservative amino acid substitutions. Also included in the present context as HPPD proteins of the present invention are HPPD proteins comprising the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, but in which 1-20, 1-15, 1-10 or 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids have been suppressed or substituted by other amino acids, particularly a protein that retains the enzymatic activity of HPPDs and confers tolerance to herbicides that inhibit HPPD when expressed in a host plant. HPPD proteins encoded by DNA sequences homologous to the DNA sequences of the invention are described in the present context as described below, or HPPD proteins encoded by a DNA sequence that hybridizes with at least a portion. (of at least 20-30 nucleotides) of the DNA of SEQ ID NO. 1, or which is obtainable using a primer based on SEQ ID NO. 1, or HPPD proteins with identity between sequences of at least 75% with SEQ ID NO. 4, which are encoded by a DNA sequence found in the genome sequence of a microorganism, such as a bacterium, such as a microorganism of the Kordia genus. Included in the present context as an HPPD protein of the present invention is a Kordia HPPD protein that confers herbicide tolerance to plants when expressed in such plants, wherein said tolerance is to an HPPD inhibitory agent. such as mesotrione, tembotrione, isoxaflutol or bicyclopyrone, particularly said HPPD protein is a Kordia algicide HPPD protein, such as a protein comprising the sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387. This includes mutant or variant HPPD proteins as described below.

The present disclosure includes and provides an antibody capable of specifically binding a substantially purified protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 5, 6 or 7, or sequences derived therefrom according to with an amino acid replacement as described in one or more of Tables (i), (ii) or (iii) above.

A further aspect of the disclosure concerns antibodies, single-chain antigen-binding molecules, or other proteins that specifically bind to one or more of the protein or peptide molecules of the invention and their homologous compounds, fusions or fragments. In a particularly preferred embodiment, the antibody specifically binds to a protein having the amino acid sequence set forth in SEQ ID NO: 4-7 or a fragment thereof, or sequences derived therefrom in accordance with a replacement. of amino acids as described in one or more of Tables (i), (ii) or (iii) above.

In another disclosure the antibody specifically binds to a fusion protein comprising an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 4-7, or a fragment thereof, or sequences derived therefrom. same according to an amino acid replacement as described in one or more of Tables (i), (ii) or (iii) above.

In another disclosure, the antibody specifically binds to a fusion protein comprising an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 4-7, or a fragment thereof, or sequences derived from the same according to an amino acid replacement as described in one or more of Tables (i), (ii) or (iii) above.

Also included herein as "HPPD DNA" are DNA sequences encoding an HPPd protein of the invention whose DNA sequences have been adapted for expression in microorganisms or plants, such as by replacement of native codons with more codons. preferred in a host cell, or in which certain restriction sites have been added or removed for ease of cloning, or a DNA sequence with a certain number of nucleotides added, replaced or deleted. This also includes isolated DNA sequences and variant, mutant or synthetic DNA or nucleic acids, as described below. In a particular embodiment, the Kordia HPPD DNA of the present invention is expressed in plants under the control of a promoter that allows the expression of exogenous genes in plants. In a further particular embodiment, next to the N-terminal end of the HPPD enzyme expressed in this manner is located a signal peptide, such as a transit peptide, preferably a plastid transit peptide, such as a transit peptide. chloroplasts with about 120 amino acids (from about 30 to about 120 amino acids) most preferably a double transit peptide, such as an optimized transit peptide whose first part originates from sunflower (Helianthus annuus) and whose second part originates from Zea mays (corn) (which has been described in US Patent 5,188,642) or a plastid transit peptide that originates from the small subunit of ribulose biscarboxylase / oxygenase from plants (RuBisCO ssu, acronym for ribulose biscarboxylase / oxygenase) small subunit), which when appropriate includes a few

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amino acids of the N-terminal part of the mature RuBisCO ssu (EP 189 707).

In a further particular embodiment, the present invention includes a DNA encoding an HPPD protein of the present invention that is derived or obtainable from SEQ ID NO. 1 and is optimized for expression in E. coli, such as a codon optimized DNA, for example a DNA comprising the sequence of SEQ ID NO. 2 from nucleotide position 25 to nucleotide position 1,182 (which includes defined positions).

In a further particular embodiment, the present invention includes a DNA encoding an HPPD protein of the present invention that is derived from sEq ID NO. 1 and is optimized for expression in plants, such as a codon optimized DNA, for example a DNA comprising the sequence of SEQ ID NO. 3 from nucleotide position 400 to nucleotide position 1,557 (which includes defined positions).

In a further particular embodiment, the HPPD of the invention, such as the HPPD comprising the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387, or the HPPD comprising the amino acid sequence of any one of SEQ ID NO. 4 to 7, it is less sensitive than the endogenous HPPD of the host plant to an inhibitor herbicide of the HPPD of the isoxazole class, dikettonitriles, tricetonas or pyrazolinates, or to an HPPD inhibitor herbicide, selected from isoxaflutol, tembotrione, mesotrione , sulcotrione, pyrasulfotol, topramezone, 2-cyano-3-cyclopropyl-1- (2-SO2CH3-4-CF3phenyl) - propane-1,3-dione and 2-cyano-3-cyclopropyl-1- (2-SO2CH3- 4-2.3 Ch phenyl) -propane-1,3-dione, bicyclopyrone,

benzobiciclone, tefuriltrione and pyrazoxyphene.

In a further particular disclosure, the present invention includes a DNA encoding an HPPD protein of the present invention that is derived from SEQ ID NO. 1 and is optimized for expression in E. coli, such as a codon optimized DNA, for example a DNA comprising the sequence of SEQ ID NO. 2 from nucleotide position 25 to nucleotide position 1,182 (which includes defined positions) encoding a less sensitive HPPD than the endogenous HPPD of the host plant to at least one HPPD inhibitor herbicide of the isoxazole class, dicetonitriles, tricetonas or pyrazolinates, preferably to trembling, mesotrione, bicyclopyrone, tefuriltrione, isoxaflutol, dicetonitrile, pyrasulfotol, topramezone, sulcotrione, pyrazolate and benzofenap.

In a further particular embodiment, the present invention includes a DNA encoding an HPPD protein of the present invention that is optimized for expression in plants, such as a codon optimized DNA, for example a DNA comprising the sequence of SEQ ID No. 3 from nucleotide position 400 to nucleotide position 1,557 (which includes defined positions), which encodes an HPPD less sensitive than the endogenous HPPD of the host plant to at least one herbicide inhibitor of class HPPD of the isoxazoles, dicetonitriles, tricetonas or pyrazolinates, preferably to trembling, mesotrione, bicyclopirone, tefuriltrione, isoxaflutol, dicetonitrile, pyrasulfotol, topramezone, sulcotrione, pyrazolate and benzofenap.

In a further particular embodiment, the present invention relates to plants, parts of plants, plant cells and progeny of these plants comprising a DNA encoding an HPPd protein of the invention, which is optimized for expression in E. coli , or is optimized for expression in plants, such as a codon optimized DNA, for example a DNA comprising the sequence of SEQ ID NO. 2 from nucleotide position 25 to nucleotide position 1,182 (which includes defined positions) or SEQ ID NO. 3 from nucleotide position 400 to nucleotide position 1,557 (which includes defined positions) encoding a less sensitive HPPD than the endogenous HPPD of the host plant. Such plants include, but are not limited to, plants grown in the field, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, corn, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, soybean species, sugar cane, peas, beans, poplar, grape, citrus fruits, alfalfa, rye, oats, grass and forage grasses, flax and oilseed rapeseed, and nut-producing plants.

In a more particular embodiment, the present invention relates to plants, parts of plants, plant cells and progeny of these plants comprising any of the DNAs encoding an HPPD of the invention that is optimized for expression in E. coli, or optimized for expression in plants, such as a codon optimized DNA, for example a DNA comprising the sequence of SEQ ID NO. 2 from nucleotide position 25 to nucleotide position 1,182 (which includes defined positions) or SEQ ID NO. 3 from nucleotide position 400 to nucleotide position 1,557 (which includes defined positions) encoding a less sensitive HPPD than the endogenous HPPD of the host plant, and in which the plants are selected from the group consisting of canola, sunflower, tobacco, sugar beet, cotton, corn, wheat, barley, rice, potato, soybean species, sugar cane, peas, beans, poplar, grape, alfalfa, rye, oats, grass and forage grasses, flax and rapeseed of oilseed, and nut-producing plants, even more preferably such plants are selected from the group consisting of species of soybeans, rice, sugar beets, wheat, cotton, canola, oilseed rape or corn.

In another particular embodiment, the HPPD protein of the invention comprises the sequence of SEQ ID NO. 7 and is less sensitive to an HPPD inhibitor agent of the tricetonas class (called tricetone HPPD inhibiting agents), such as tremorotrione, mesotrione sulcotrione, bicyclopirone, tefuriltrione, particularly trembling, or of the class of dicetonitriles (isoxaflutol) or of the pyrazolinate class (which

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they are called HPPD pyrazolinate inhibitors), such as pyrasulfotol, pyrazolate, topramezone, benzophenap compared to the endogenous non-mutated HPPD of a plant, particularly the host plant in which said HPPD of the invention is expressed or is to be expressed.

The enzymatic activity of HPPD proteins can be measured by any procedure that makes it possible either to measure the decrease in the amount of HPP or O2 substrates, or to measure the accumulation of any of the products derived from the enzymatic reaction. say homogentisate or CO2. In particular, the activity of HPPD can be measured by the procedure described in the quotations of Garcia et al. (1997), Biochem. J. 325, 761-769 or Garcia et al. (1999), Plant Physiol. 119, 1507-1516, which are incorporated in the present context by reference.

According to the invention, an HPPD inhibitor agent of the tricetonas class (or which is called tricetone HPPD inhibitor agent) means an HPPD inhibitor agent having a tricetone skeleton. As an example of such tricetone HPPD inhibiting agents, mention may be made of the molecules sulcotrione [ie 2- [2-chloro-4- (methylsulfonyl) -benzoyl] -1,3-cyclohexanedione], mesotrione [ie 2- [4- (methylsulfonyl) -2-nitrobenzoyl] -1,3-cyclohexanedione], and trembling [ie 2- [2-chloro-4- (methylsulfonyl) -3 - [(2,2,2-trifluoroethoxy) methyl ] benzoyl] -1,3-cyclohexanedione], tefuryltrione [ie 2- {2-chloro-4-mesyl-3 - [(RS) -tetrahydro-2- furylmethoxymethyl] -benzoyl} cyclohexane-1,3-dione] , bicyclopyrone [ie 4-hydroxy-3- {2 - [(2-methoxyethoxy) methyl] -6- (trifluoromethyl) -3-pyridylcarbonyl} bicyclo [3.2.1] oct-3-en-2-one], benzobicyclone [ie 3- (2-chloro-4-mesylbenzoyl) -2-phenylthiobicyclo [3.2.1] oct-2-en-4-one].

According to the invention, an HPPD inhibitor agent of the pyrazolinate class (or HPPD pyrazolinate inhibitor agent) means an HPPD inhibitor that has a pyrazole radical. As an example of such HPPD pyrazolinate inhibitor agents, mention may be made of topramezone molecules [ie [3- (4,5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl] - (5 -hydroxy-1-methyl-1H-pyrazol-4-yl) methanone] and pyrasulfotol [(5-hydroxy-1,3-dimethylpyrazol-4-yl- (2-mesyl-4-trifluoromethylphenyl) methanone].

The present invention also relates to a nucleic acid sequence, particularly an isolated DNA, preferably a plant-expressible chimeric gene, which encodes the Kordia HPPd of the invention and adapted sequences thereof.

The present invention also relates to a nucleic acid sequence encoding an HPPD enzyme of the present invention that retains its properties of catalyzing the conversion of para-hydroxyphenylpyruvate into homogentisate and that is less sensitive to HPPD inhibitors of the class. of tricetonas such as tremorotrione, sulcotrione and mesotrione, or of the class of pyrazolinates such as pyrasulfotol and topramezone, tefuriltrione, bicyclopirone, benzobicyclone that endogenous non-mutated plant HPPD, and whose amino acid encoded sequence shows an identity between sequences with SEQ ID NO. 4 of at least 75%, 80%, particularly at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99%

In a more particular embodiment, the nucleic acid sequence of the invention encodes an HPPD enzyme that is less sensitive to an HPPD inhibitor agent of the tricetonas class, such as tremorotrione, sulcotrione, mesotrione, bicyclopyrone and tefuriltrione, of the class of isoxazoles such as isoxaflutol from the pyrazolinate class (which are called HPPD pyrazolinate inhibitors), such as pyrasulfotol, pyrazolate, topramezone, benzofenap, or from the class of diketones such as dicetonitrile, which endogenous HPPD from The host plant.

In accordance with the present invention, it is understood that a "nucleic acid sequence" is a nucleotide sequence that may be of the DNA or RNA type, preferably of the DNA type, and in particular double stranded, whether of origin natural or synthetic, in particular a DNA sequence in which the codons encoding the HPPD according to the invention have been optimized according to the host organism in which it is to be expressed (for example, replacing codons with those codons that are more preferred or highly preferred in treatment tables with codons of said host organism or the group to which said host organism belongs, as compared to the original or source organism).

An "isolated nucleic acid / DNA / protein," as used herein, refers to a nucleic acid / DNA / protein that is not naturally occurring (such as an artificial or synthetic DNA with a nucleotide sequence). which is different from that of naturally occurring DNA, or a modified protein) or that is no longer in the natural environment in which it was originally present, for example a sequence encoding a DNA, associated with a regulatory element heterologous (such as a bacterial coding sequence operatively connected to an expressible promoter in a plant) in a chimeric gene, a DNA transferred into another host cell, such as a transgenic plant cell.

In view of a particular embodiment of the invention and of the solution sought, that is to say an HPPD that is less sensitive to an inhibitor of the HPPD of tricetone, isoxazole or pyrazolinate, the tolerance level measurement is analyzed using the procedure widely described. in WO 2009/14407 as described below, using an HPPD inhibitor of tricetone, isoxazole or pyrazolinate, particularly an HPPD inhibitor selected from trembling, mesotrione, pyrasulfotol, topramezone sulcotrione, bicyclopirone, dicetonitrile, benzophenap , pyrazolate and tefuriltrione.

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The terminology of a DNA or a protein "comprising" a certain "X" sequence, as used throughout the text, refers to a DNA or a protein that includes or contains at least the "X" sequence of such that other nucleotide or amino acid sequences may be included next to the 5 '(or N-terminal) and / or 3' (or C-terminal) ends, for example (the nucleotide sequence of) a selectable marker protein , (the nucleotide sequence of) a transit peptide, and / or a 5 'leader sequence or a 3' trailer sequence. Similarly, it should be understood that the use of the term "understand", "which comprises" or "comprises" throughout the text and the claims of this application implies the inclusion of a designated whole part or stage or a set set of whole parts or stages, but not the exclusion of any other whole part or stage or set of whole parts or stages. The coding regions encoding an HPPD comprise a nucleotide sequence that encodes proteins with the amino acid sequences set forth in SEQ ID NO 4, 5, 6 and 7, such as the nucleotide sequences of SEQ ID NO 1, 2 and 3

However, it will be apparent that variants of these nucleotide sequences, including insertions, deletions and substitutions thereof, can also be used with the same effect. Similarly, sequences homologous to the aforementioned nucleotide sequences, from different species of Kordia algicide, can be used.

The variants of the nucleotide sequence described above will have an identity between sequences that will preferably be at least about 70%, 80%, 85% or 90% or at least 95% with identified nucleotide sequences encoding HPPD enzymes such as those identified in the sequence list.

A protein that has "substantially the same amino acid sequence" as a protein as described herein, refers to a protein that has an identity between sequences of at least 90%, particularly at least 95%, preferably at least 97% with a protein according to the invention, in which the percentage of identity between sequences is determined using the blosum62 rating matrix in the GAP program of the GCG Wisconsin package (Madison, Wisconsin, USA). .) version 10.0 (GCG defects were used). "Identity between sequences", as used throughout this application, when referring to proteins, refers to the percentage of identical amino acids when using this specified analysis. "Sequence identity", as used herein, when referring to DNA sequences, is determined using the nwsgapdna rating matrix in the GAP program of the GCG Wisconsin package (Madison, Wisconsin, USA). ) version 10.0 (GCG defects were used).

For the purpose of the present invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two sequences, optimally aligned, having identical residues ( x 100) divided by the number of positions that have been compared. A hole, that is to say a position in an alignment in which one remainder is present in one sequence but not in the other, is considered a position with non-identical remains. The alignment of the two sequences is done by the algorithm of Needleman and Wunsch (Needleman and Wunsch 1970). The above computer-assisted sequence alignment can be conveniently performed using a classic software program, such as the GAP that is part of the Wisconsin Package Version 10.1 package (from Genetics Computer Group, Madison, Wisconsin, USA) using the default rating matrix with a penalty for creating a gap of 50 and a penalty for prolongation of a gap of 3.

Nucleotide sequences homologous to nucleotide sequences encoding an HPPD enzyme according to the invention can be identified by an in silico analysis of genomic sequence data.

A homologous nucleotide sequence can also be identified and isolated by hybridization under stringent conditions using as identified probes nucleotide sequences encoding HPPD enzymes according to the invention or parts thereof. Said parts should preferably have a nucleotide sequence comprising at least 40 consecutive nucleotides from the coding region of gene sequences encoding the HPPD according to the invention, preferably from the coding region of SEQ ID NO. 1, SEQ ID NO. 2nd SEQ ID NO. 3. However, the probes may comprise longer regions of nucleotide sequences derived from the nucleic acids encoding the HPPDs, such as approximately 50, 60, 75, 100, 200 or 500 consecutive nucleotides from any of the genes of HPPD that have been mentioned. Preferably, the probe should comprise a nucleotide sequence that encodes a highly conserved region, which can be identified by alignment of the different HPPD proteins.

"Rigorous hybridization conditions" as used herein means that a hybridization will generally be performed if there is an identity between sequences of at least 95% and preferably at least 97% between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt solution , 10% dextran sulfate and 20 pg / ml of a cut and denatured carrier DNA such as salmon sperm DNA, followed by a wash of the hybridization support in 0.1xSSC at about 65 ° C, preferably twice during approximately 10 minutes Other hybridization and washing conditions are well known and are given as an example in the quotation of Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY (1989 ), particularly chapter 11.

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Such variant sequences can also be obtained by DNA amplification using oligonucleotides specific for HPPD genes encoding enzymes as primers, such as, but not limited to, oligonucleotides comprising from about 20 to about 50 consecutive nucleotides selected from the sequences. nucleotide of SEQ ID NO 1, 2, 3, or its complement.

The invention also comprises variant HPPD enzymes that are amino acid sequences similar to the HPPD amino acid sequence of SEQ ID NO. 4 in which one or more amino acids have been introduced, deleted or substituted. In the present context, the variants of an amino acid sequence refer to those polypeptides, enzymes or proteins that have a catalytic activity similar to that of the amino acid sequences described herein, despite any substitutions, additions or amino acid deletions made in them. Preferably, the amino acid variant sequence has an identity between sequences of at least about 80%, or 85 or 90% or 95% with the amino acid sequence of SEQ ID NO. 4. Also preferably, a polypeptide comprising the amino acid variant sequence has the enzymatic activity of the HPPD. Methods for determining the enzymatic activity of HPPDs are well known in the art and include assays that are described extensively in WO 2009/144079 or in WO 2002/046387.

The substitutions include amino acid alterations in which an amino acid is replaced by a different amino acid residue of a natural or an unconventional origin. Such substitutions can be classified as "conservative" when an amino acid residue contained in an HPPD protein of the present invention is replaced by another naturally occurring amino acid that has a similar character, for example Gly ^ Ala, Val ^ Ile ^ - Leu, Asp ^ -Glu, Lys ^ Arg, Asn ^ Gln or Phe ^ -Trp ^ Tyr. The substitutions comprised by the present invention may also be "non-conservative", when an amino acid residue that is present in an HPPD protein of the present invention is replaced by an amino acid having different properties, such as a naturally occurring amino acid from from a different group (for example by substitution of a charged or hydrophobic amino acid with alanine). Amino acid substitutions are typically single residues, but may also be multiple residues, either clustered or dispersed. The amino acid deletions will usually be of the order of approximately 1-10 amino acid residues, while the inserts can be of any length. Deletions and insertions can be made at the N-terminal end, at the C-terminal end, or they can be internal deletions or insertions. Generally, inserts within the amino acid sequence will be smaller than fusions at the amino or carboxy terminals and of the order of 1 to 4 amino acid residues. "Similar amino acids," as used herein, refers to amino acids that have similar chains of amino acids, that is, amino acids that have polar, non-polar, or practically neutral side chains. "Non-similar amino acids," as used in the present context, refers to amino acids that have different amino acid side chains, for example an amino acid with a polar side chain is not similar to an amino acid with a non-polar side chain. Polar side chains usually tend to be present on the surface of a protein when they can interact with the aqueous environment found in cells ("hydrophilic" amino acids). On the other hand, "non-polar" amino acids tend to reside within the center of the protein where they can interact with similar non-polar neighbors ("hydrophobic" amino acids). Examples of amino acids that have polar side chains are arginine, asparagine, aspartate, cysteine, glutamine, glutamate, histidine, lysine, serine and threonine (all of them hydrophilic, except for the cysteine that is hydrophobic). Examples of amino acids that have polar side chains are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline and tryptophan (all of them hydrophobic, except glycine which is neutral).

Antibodies that specifically recognize an HPPD enzyme according to the invention are also understood by the present invention.

The invention also relates to the use, in a process for transforming plants, of a nucleic acid encoding an HPPD according to the invention as a marker gene or as a coding sequence that makes it possible to confer to the plant a tolerance to herbicides that are HPPD inhibitors, and the use of HPPD inhibitors in plants comprising a nucleic acid sequence encoding an HPPD according to the invention. In one embodiment of the present invention, in said use the HPPD inhibiting agents are tricetonas or pyrazolinates, preferably trembling, mesotrione or sulcotrione, bicyclopyrone and tefuriltrione. Of course, it is understood that this sequence can also be used in combination with (a) marker or markers of genes and / or different sequence or sequences that encode or encode one or more proteins with useful agricultural properties.

In the commercial production of cultivated plants, it is desirable, within a reliable administration of pesticides, to eliminate unwanted plants (ie "weeds") from a field of cultivated plants. An ideal treatment would be one that could be applied to a complete field, but that would only eliminate unwanted plants while leaving cultivated plants unaffected. One such treatment system could involve the use of cultivated plants that are tolerant to a herbicide such that when the herbicide is sprayed on a field of cultivated plants tolerant to herbicides, the cultivated plants will continue to thrive while non-tolerant weeds to Herbicides were annihilated or severely damaged. Ideally, said treatment systems will take advantage of variable herbicidal properties such that repression of

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Weeds could provide the best possible combination of flexibility and economy. For example, individual herbicides have different longevity in the field, and some herbicides persist and are effective for a relatively long period of time after they have been applied to a field, while other herbicides are rapidly decomposed to give other different compounds. and / or not active. An ideal treatment system could allow the use of different herbicides in such a way that breeders could select the choice of herbicides for a particular situation as desired.

While a number of herbicide-tolerant cultivated plants are currently commercially available, an issue that has emerged for many commercial herbicides and combinations of herbicides and cultivated plants, is that individual herbicides typically have an incomplete spectrum of activities against species. of current weeds. For most of the individual herbicides that have been in use for some time, populations of weed species and herbicide-resistant biotypes have been more prevalent (see for example, Tranel and Wright (2002) Weed Science 50: 700-712 ; Owen and Zelaya (2005) Pest Manag. Sci. 61: 301-311). Transgenic plants that are resistant to more than one herbicide have been described (see, for example, W02005 / 012515). However, improvements are continually being demanded in any aspect of the production of cultivated plants, weed repression options, prolongation of the repression of residual weeds, and an improvement in the yield of cultivated plants.

The protein or HPPD gene of the invention is advantageously combined in plants with other genes encoding proteins or RNA that confer useful agronomic properties to said plants. Among the genes that encode proteins or RNA that confer useful agronomic properties to transformed plants, mention can be made of DNA sequences encoding proteins that confer tolerance to one or more herbicides that, according to their chemical structure, differ from herbicides inhibitors of HPPD, and others that confer tolerance to certain insects, of which they confer tolerance to certain diseases, DNA encoding RNA that provide repression of nematodes or insects.

Such genes are described in particular in published PCT patent applications WO 91/02071 and WO95 / 06128.

Among the DNA sequences encoding proteins that confer tolerance to certain herbicides in plant cells and transformed plants, mention may be made of a bar or PAT gene or of the Streptomyces coelicolor gene described in WO2009. / 152359 that confers herbicide tolerance, a gene that encodes an appropriate EPSPS (acronym for Enol Pyruvyl Shikimate Phosphate Syntase, enolpiruvilshikimato phosphate synthase) that confers tolerance to herbicides that have an EPSPS as a target, such as glyphosate and its salts (US documents 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US 5,627,061, US 5,633,435), or a gene encoding a glyphosate oxidoreductase (US 5,463,175).

Among the DNA sequences encoding an appropriate EPSPS that confer tolerance to herbicides that have an EPSPS as a target, mention will be made more particularly of the gene encoding a plant EPSPS, in particular a corn EPSPS, particularly a corn EPSPS comprising two mutations, particularly a mutation at amino acid position 102 and a mutation at amino acid position 106 (WO 2004/074443), and which is described in US patent application 6566587, hereinafter referred to as This EPSPS double mutant corn or 2mEPSPS document, or of the gene encoding an EPSPS isolated from Agrobacterium and which is described by SEQ ID NO. 2 and SEQ ID NO. 3 of US Patent 5,633,435, which is also called CP4.

Among the DNA sequences encoding an appropriate EPSPS that confer tolerance to herbicides that have an EPSPS as a target, mention will be made more particularly of the gene encoding an EPSPS GRG23 from Arthrobacter globiformis, but also the GRG23 ACE1, GRG23 ACE2 mutants , or GRG23 ACE3, particularly the GRG23 mutants or variants as described in WO2008 / 100353, such as GRG23 (ace3) R173K of SEQ ID NO. 29 in WO2008 / 100353.

In the case of DNA sequences encoding an EPSPS, and more particularly encoding the above genes, the sequence encoding these enzymes is advantageously preceded by a sequence encoding a transit peptide, in particular the "optimized transit peptide" which is described in US 5,510,471 or 5,633,448.

In WO 2007/024782, plants are described which are glyphosate tolerant and at least one ALS inhibitor (acetolactate synthase). More specifically, plants containing genes encoding GAT polypeptide (Glyphosate-N-Acetyl Transferase) and a polypeptide that confers resistance to ALS inhibiting agents are described.

In US 6,855,533, transgenic tobacco plants containing mutated Arabidopsis ALS / AHAS genes were described.

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In US Pat. No. 6,153,401, plants are described which contain genes encoding 2,4-D monooxygenases that confer tolerance to 2,4-D (2,4-didorophenoxyacetic acid) by metabolization.

In US 2008/0119361 and US 2008/0120739, plants containing genes encoding dicamba monooxygenases that confer tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) by metabolization are described.

All herbicide tolerance traits, mentioned above, can be combined with those that develop a tolerance to HPPDs that are subject matter of the present invention.

Among the DNA sequences encoding proteins that concern insect tolerance properties, mention will be made more particularly of the Bt proteins widely described in the literature and well known to those skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorhabdus (WO 97/17432 & WO 98/08932).

Among such DNA sequences encoding proteins of interest that confer new insect tolerance properties, mention will be made more particularly of the Bt Cry or VIP proteins, widely described in the literature and well known to those skilled in the art. These include the Cry1F protein or hybrids derived from a Cry1F protein (for example, the Cry1A-Cry1F hybrid proteins described in US 6,326,169; US 6,281,016; US 6,218,188, or toxic fragments thereof) , the Cry1A type proteins or toxic fragments thereof, preferably the Cry1Ac protein or hybrids derived from the Cry1Ac protein (for example, the Cry1Ab-Cry1Ac hybrid protein described in US 5,880,275) or the Cry1Ab protein or Bt2 or insecticidal fragments thereof as described in EP451878, Cry2Ae, Cry2Af or Cry2Ag proteins as described in WO02 / 057664 or toxic fragments thereof, the Cry1A.105 protein described therein WO 2007/140256 (SEQ ID NO. 7) or a toxic fragment thereof, the VIP3Aa19 protein with the NCBI accession number ABG20428, the VIP3Aa20 protein with the NCBI accession number ABG20429 (SEQ ID NO. 2 in the do WO 2007/142840), VIP3A proteins produced during the events in COT202 or COT203 cotton (WO 2005/054479 and WO 2005/054480, respectively), the Cry proteins described in WO01 / 47952, the VIP3Aa protein or a toxic fragment thereof, as described as described in the quotation of Estruch et al. (1996), Proc Natl Acad Sci USA. 28; 93 (11): 5389-94 and in US 6,291,156, insecticidal proteins from Xenorhabdus (as described in WO98 / 50427), from Serratia (particularly from S. entomophila) or from Strains of Photorhabdus species, such as Tc proteins from Photorhabdus as described in WO98 / 08932 (for example, in the citations of Watercamp et al., 2001, Appl Environ Microbiol. 67 (11): 5017-24 ; and from Ffrench-Constant and Bowen, 2000, Cell Mol Life Sci .; 57 (5): 828-33). Also included in the present context are any variants or mutants of any of these proteins that differ in some (1-10, preferably 1-5) amino acids from any of the above sequences, particularly the sequence of their toxic fragment, or which they are fused with a transit peptide, such as a plastid transit peptide, or other / or protein or peptide.

The present invention also relates to a chimeric gene (or expression cassette) comprising a coding sequence as well as the heterologous regulatory elements in the 5 'and / or 3' positions, at least in the 5 'position, which are capable of function within a host organism, in particular of plant or plant cells, with the coding sequence containing at least one nucleic acid sequence encoding an HPPD as defined previously.

In a particular embodiment, the present invention relates to a chimeric gene as previously described, in which the host organism is selected from bacteria, yeasts, Pichia, fungi, baculovirus, in vitro cells, protoplasts, plant cells, plants , parts of plants, and seeds of these plants.

In another particular embodiment, the present invention relates to a chimeric gene as previously described, in which the chimeric gene contains, at the 5 'position of the nucleic acid sequence encoding an HPPD according to the invention, a sequence of nucleic acid encoding a plant transit peptide, this sequence being arranged between the promoter region and the sequence encoding the HPPD according to the invention such that the expression of a transit peptide fusion protein is allowed / HPPD.

In a further particular embodiment, the present invention relates to the use of HPPD inhibitor herbicides in plants, parts of plants or plant seeds comprising an HPPD tolerant gene according to the invention, or to the use of herbicide inhibitors of HPPD in a land in which said plants, parts of plants or seeds are to be grown or sown, either alone or in combination with one or more other known herbicides that act differently from inhibitors of the HPPD. In a more particular embodiment, the HPPD inhibitor herbicide that is employed is selected from the group consisting of tricetons (which are called tricetonic HPPD inhibitors), such as trembling, sulcotrione, mesotrione, bicyclopirone, tefuriltrione, particularly trembling, in the class of diketones such as dikettonitriles of the isoxazole class such as isoxaflutol or in the class of pyrazolinates (which are called HPPD pyrazolinate inhibitors), such as pyrasulfotol, pyrazolate, topramezone, benzofenap; Even more specifically, the present invention relates to the application of trembling, mesotrione, dicetonitrile,

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bicyclopyrone, tefuriltrione, benzophenap, pyrasulfotol, pyrazolate and sulcotrione to said plants, parts of plants or plant seeds that are tolerant to HPPD inhibitory agents.

As a regulatory sequence that functions as a promoter in plant and plant cells, use may be made of any promoter sequence of a gene that is naturally expressed in plants, in particular of a promoter that is expressed especially in leaves. of plants, such as for example "constitutive" promoters of bacterial, viral or plant origin, or of "light-dependent" promoters, such as that of a small subunit gene of ribulose biscarboxylase / plant oxygenase (RuBisCO) or of any appropriate known gene expressible by promoters, which may be used. Among promoters of plant origin, mention will be made of histone promoters as described in EP 0 507 698 A1, the rice actin promoter (US 5,641,876), or a plant ubiquitin promoter (US document 5,510,474). Among the promoters of a plant virus gene, mention will be made of those of the cauliflower mosaic virus (CaMV 19S or 35S, Sanders et al. (1987), Nucleic Acids Res. 15 (4): 1543-58.) , those of the circovirus (Australian patent document AU 689 311) or those of the Cassava vein mosaic virus (CsVMV, US 7,053,205).

In one embodiment of the present invention, a specific promoter sequence can be used for particular regions or tissues of plants in order to express the HPPD proteins of the invention, such as seed-specific promoters (Datla, R. et al. , 1997, Biotechnology Ann. Rev. 3, 269-296), especially the napine promoter (EP 255 378 A1), the phaseolin promoter, the glutenin promoter, the heliantinin promoter (WO 92/17580), the albumin promoter (WO 98/45460), the oleosin promoter (WO 98/45461), the SAT1 promoter or the SAT3 promoter (PCT / US98 / 06978). It is also possible to make use of an inducible promoter advantageously chosen among the phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), gene promoter promoters PR1 family of nopaline synthase (nos) and vspB (US 5 670 349, Table 3), the HMG2 promoter (US 5 670 349), the apple beta-galactosidase promoter (ABG1) and the promoter of Apple aminocyclopropane carboxylate synthase (ACC synthase) (WO 98/45445).

According to the invention, it is also possible to use, in combination with the promoter, other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators ("enhancers"), for example the translation activator of tobacco mosaic virus (TMV), which is described in application WO 87/07644, or tobacco corrosion virus (TEV) which has been described by Carrington & Freed 1990, J. Virol. 64: 1590-1597, for example, or introns such as corn adh1 intron or rice actin intron 1.

In a further particular disclosure the gene of the invention is present in plants in multiple, preferably two copies, each controlled by a different plant expressible promoter.

In a further particular embodiment, the chimeric gene of the invention can be combined with any additional chimeric gene encoding an HPPD protein, preferably those different genes are controlled by different regulatory elements being active in plants.

In a further particular disclosure the chimeric gene of the invention can be combined with a CYP450 corn monooxygenase gene (nsf1 gene) being under the control of an identical or different plant expressible promoter.

As a terminating or polyadenylation terminator sequence, any corresponding sequence of bacterial origin can be used, such as that of the Agrobacterium tumefaciens nos terminator, of viral origin, such as that of the CaMV 35S terminator, or of plant origin, such as that of a histone terminator as described in published patent application EP 0 633 317 A1.

The term "gene" as used herein refers to a region that encodes a DNA flanked by 5 'and / or 3' regulatory sequences, which allow an RNA to be transcribed that can be translated into a protein, which typically comprises at least one promoter region. A "chimeric gene," when referring to a DNA encoding an HPPD of the present invention, refers to a sequence of a DNA encoding an HPPD that has 5 'and / or 3' regulatory sequences different from the sequences. 5 'and / or 3' bacterial regulators present in nature that drive the expression of the HPPD protein in its natural host cell (also referred to as "heterologous promoter" or "heterologous regulatory sequences").

The terms "DNA / protein comprising sequence X" and "DNA / protein with sequence comprising sequence X", as used herein, refer to a DNA or a protein that includes or contains at least the X sequence in its nucleotide or amino acid sequence, such that other nucleotide or amino acid sequences can be included next to the 5 '(or N-terminal) and / or 3' (or C-terminal) end, for example , a transit or N-terminal signal peptide. The term "comprising", as used in the present context, is an open-ended language modality in the meaning of "include," meaning that elements other than those specifically cited may also be present. The term "consisting of", as used in the present context, is a closed-end language modality, that is, only those elements that are specifically cited are present. The term "DNA encoding a

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Protein comprising the sequence X ", as used in the present context, refers to a DNA comprising a coding sequence, which after transcription and translation, results in a protein containing at least one amino acid sequence X A DNA encoding a protein does not need to be a DNA present in nature and can be a semi-synthetic, fully synthetic or artificial DNA and can include introns and flanking regions in 5 'and / or 3'. The term "nucleotide sequence" as used herein refers to the sequence of a DNA or RNA molecule, which may be in the form of a single strand (single stranded) or double stranded (double stranded).

The HPPD proteins according to the invention can be equipped with a signal peptide according to processes well known in the art, see, p. eg, published PCT patent application WO 96/10083, or may be replaced by other peptides, such as a chloroplast transit peptide (eg, Van Den Broeck et al., 1985, Nature 313, 358, or a modified chloroplast transit peptide of US 5,510,471) which causes a transport of the protein to the chloroplasts, by a secretory signal peptide or by a peptide that directs the protein to the target of other plastids, mitochondria, of the ER, or other organelles, or they can be replaced by a methionine amino acid or by a methionine-alanine dipeptide. Signal sequences for the direction to intracellular organelles or for secretion outside the plant cell or towards the cell wall, are found in proteins directed to targets or segregated naturally, preferably those described by Klosgen et al. (1989, Mol. Gen. Genet. 217, 155-161), Klosgen and Weil (1991, Mol. Gen. Genet. 225, 297-304), Neuhaus & Rogers (1998, Plant Mol. Biol. 38, 127- 144), Bih et al. (1999, J. Biol. Chem. 274, 22884-22894), Morris et al. (1999, Biochem. Biophys. Res. Commun. 255, 328-333), Hesse et al. (1989, EMBO J. 8 2453-2461), Tavladoraki et al. (1998, FEBS Lett. 426, 62-66), Terashima et al. (1999, Appl. Microbiol. Biotechnol. 52, 516-523), Park et al. (1997, J. Biol. Chem. 272, 6876-6881), Shcherban et al. (1995, Proc. Natl. Acad. Sci USA 92, 92459249), all of whose citations are incorporated in the present context by reference, particularly the signal peptide sequences from proteins targeting or segregated targets of corn, cotton, soybeans or rice A DNA sequence encoding said plant signal peptide can be introduced into the chimeric gene encoding the HPPD protein for plant expression.

Unless otherwise stated in the examples, all processes to produce and manipulate a recombinant DNA are carried out by the classical procedures described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY (1989), and in volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Classic materials and procedures for work in molecular biology are described in Plant Molecular Biology Labfax (1993) by R.R.D. Croy, published jointly by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK). Processes for PCR technology (acronym for Polymerase Chain Reaction = polymerase chain reaction) can be found in “PCR protocols: a guide to methods and applications”, compiled by MA Innis, D.H. Gelfand, J.J. Sninsky and T.J. White (Academic Press, Inc., 1990).

The terms "tolerance", "tolerant" or "less sensitive" are used interchangeably and mean the relative levels of inherent tolerance of the HPPD screened according to a visible indicator phenotype of the strain or plant transformed with a nucleic acid comprising the gene encoding the respective HPPD protein in the presence of different concentrations of the various HPPD inhibitors. Dose responses and relative shifts in the dose responses associated with these indicator phenotypes (brown formation, growth inhibition, bleaching, herbicidal effect, etc.) are conveniently expressed in terms, for example, of GR50 values. (concentration for a 50% reduction in growth) or MIC (minimum inhibitory concentration) in which increases in values correspond to increases in the inherent tolerance of the expressed HPPD, in the normal way based on damage caused to plants, symptoms of meristematic bleaching, etc., in a range of different concentrations of herbicides. These data can be expressed in terms of, for example, GR50 values derived from the dose / response curves that have "a dose" plotted on the x-axis and "a percentage of destruction", "a herbicidal effect", “Numbers of green plants that sprout”, etc., represented in the axis of the y, in which increased values of GR50 correspond to increased levels of inherent tolerance of the expressed HPPD. Herbicides can be applied properly before the outbreak or after the outbreak.

Similarly, the tolerance level of the nucleic acid or gene encoding an HPPD protein according to the invention or the HPPD protein of the invention is screened by a transgenesis, regeneration, aging and a spray test of a test plant such as a tobacco plant, or a cultivated plant such as a soybean or cotton plant, and according to these results, said plants are at least 2-4 times more tolerant to HPPD inhibiting agents, such as trembling. , mesotrione, dicetonitrile and / or bicyclopyrone, that plants that do not contain any exogenous gene that encodes an HPPD protein, or that plants that contain a gene that encodes an HPPD DNA from Arabidopsis thaliana, under the control of the same promoter that that of the HPPD DNA of the invention.

It is understood that a "host organism" or "host" is any unicellular or multicellular heterologous organism into which a nucleic acid or a chimeric gene according to the invention can be introduced in order to produce an HPPD according to the invention. organisms are, in particular, bacteria, for example of E. coli, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, Pichia, fungi, in particular Aspergillus, a baculovirus or, preferably, plant and plant cells.

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It is understood that a "plant cell," according to the invention, is any cell that derives from, or is found in, a plant, and that is capable of forming, or is part of, undifferentiated tissues, such as calluses. , differentiated tissues such as embryos, parts of plants, plants or seeds. This includes protoplasts and pollen, cultured plant cells or protoplasts that have grown in vitro, and plant cells that can be regenerated to give a complete plant.

It is understood that a "plant", according to the invention, is any differentiated multicellular organism that is capable of photosynthesis, in particular a monocot or dicot organism, more especially cultivated plants that are intended or are not intended for animal nutrition or humans, such as corn or corn grain, wheat, plants of Brassica spp. such as Brassica napus or Brassica júncea, soybean species, rice, sugar cane, beet root, tobacco, cotton, vegetable plants such as cucumber, leek, carrot, tomato, lettuce, peppers, melon, watermelon, etc. The term "transgenic plants," as used in the present context, refers to plants that comprise a foreign or heterologous gene stably introduced into their genome.

In one embodiment, the invention relates to the transformation of plants. Any promoter sequence of a gene that is naturally expressed in plants, or any hybrid or combination of gene promoter elements naturally expressed in plants, including Agrobacterium or plant virus promoters, or any promoter that is appropriate for controlling transcription of a herbicide tolerance gene in plants, can be used as the promoter sequence in the plants of the invention (called "plant expressible promoter" in the present context). Examples of such promoters expressed in plants have been described above. In an embodiment of the present invention, said plant-expressible promoters are operatively connected to a coding sequence encoding an HPPD protein of the invention to form an HPPD chimeric gene of the present invention.

According to the disclosure it is also possible to use, in combination with the promoter regulatory sequence, other regulatory sequences that are located between the promoter and the coding sequence, such as intron sequences, or transcription activators (enhancers). Examples of such appropriate regulatory sequences have been described above.

Any corresponding sequence of bacterial or viral origin, such as the terminator nos from Agrobacterium tumefaciens, or of plant origin, such as a histone terminator as described in EP 0 633 317 A1, can be used as a sequence regulator of termination of transcription (and polyadenylation).

In a particular embodiment of the invention, a nucleic acid sequence encoding a transit peptide is employed in 5 '(upstream) of the nucleic acid sequence encoding the exogenous HPPD according to the invention, this sequence being arranged. transit peptide between the promoter region and the sequence encoding the exogenous HPPD, such that the expression of a fusion protein of the transit peptide and the HPPD, such as the protein of sEq ID NO, is allowed. 6 or SEQ ID NO. 7. The transit peptide makes it possible to direct the HPPD into the plastids, more especially the chloroplasts, the fusion protein being dissociated between the transit peptide and the HPPD protein of the invention when the latter enters the plastid. The transit peptide may be a single peptide, such as an EPSPS transit peptide (described in US Patent 5,188,642) or a transit peptide of the small subunit of ribulose biscarboxylase / plant oxygenase (RuBisCO ssu) which, when appropriate, includes a few amino acids from the N-terminal part of the mature sBu RuBisCO (EP 189 707 A1), or it may be a fusion of several transit peptides such as a transit peptide comprising a first transit peptide of a plant that is fused with a part of the N-terminal sequence of a mature protein that has a plastid location, this part being in turn fused with a second transit peptide of a plant, such as EP 508 909 A1 is described in patent document and, more especially, the optimized transit peptide comprising a sunflower RuBisCO ssu transit peptide fused with 22 amino acids of The N-terminal end of the RuBisCO ssu of corn, in turn fused with the transit peptide of the RuBisCO ssu of corn, as described, with its coding sequence, in patent document EP 508 909 A1. The present invention also relates to the fusion protein of a transit peptide and an HPPD and a nucleic acid or a plant-expressible chimeric gene encoding said fusion protein, in which the two elements of this fusion protein are such as have been described above.

The present invention also relates to a cloning, transformation and / or expression vector, whose vector contains at least one chimeric gene as defined above. In addition to the previous chimeric gene, this vector may contain an origin of replication.

This vector may be a plasmid or a portion of a plasmid, a cosmid, or a bacteriophage or a virus that has been transformed by introduction of the chimeric gene according to the invention. Transformation vectors are well known to the skilled person and have been widely described in the literature. The transformation vector that can be used, in particular, to transform plant or plant cells can be a virus, which can be used to transform plant or plant cells and which additionally contains its own replication and expression elements. According to the invention, the vector to transform plant cells or

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plants, is preferably a plasmid such as an unarmed Ti plasmid of Agrobacterium.

The present disclosure also relates to host organisms, in particular to the cells of plants, seeds or plants comprising a chimeric gene which in turn comprises a sequence encoding an HPPD protein of the invention, such as a protein that encodes the amino acid sequence of SEQ ID NO 4, 5, 6 or 7, as defined above, and the use of the plants or seeds of the invention in a field to grow a cultivated plant and harvest a vegetable product , for example, soybean species, rice grains, wheat, barley or corn or cotton capsules, in which, in one embodiment, said use implies the application of an HPPD inhibitor herbicide to said plants to suppress weeds. In one embodiment of the present invention, in said use, the HPPD inhibiting agents are tricetonas or pyrazolinates, preferably tremorotrione, mesotrione, topramezone or sulcotrione, bicyclopyrone, pyrasulfotol, pyrazolate, benzofenap and tefuriltrione, particularly tremorotrione.

Therefore, the present invention relates to a host organism, in particular a plant cell, a seed or a plant, which is characterized in that it contains at least one chimeric HPPD gene as previously described, or less an HPPD nucleic acid sequence as described above.

In a particular embodiment, the present invention relates to a plant cell or a plant, which is characterized in that it contains at least one nucleic acid sequence encoding an HPPD protein of the present invention, which retains its properties of catalyzing the conversion of a para-hydroxyphenylpyruvate into a homogentisate and which makes the plant more tolerant, than plants of the same species that do not comprise said HPPD protein of the present invention, particularly tricetonas, or pyrazolinates, preferably trembling, mesotrione, topramezone or sulcotrione, bicyclopyrone, pyrasulfotol, pyrazolate, benzofenap and tefuriltrione, particularly trembling, and such plants containing the HPPD of the invention have an agronomically acceptable tolerance to an inhibitor herbicide of the HPPD, particularly to tricetonas, or pyrazolonates, preferably trembling, mesotrione , topramezone or sulcotrione, bicyclopyrone, pirasu lfotol, pyrazolate, benzofenap and tefuriltrione, particularly trembling.

In another particular embodiment, the present invention relates to a plant cell or a plant characterized in that it contains at least one nucleic acid sequence encoding an HPPD of the present invention, which retains its properties of catalyzing the conversion of a para- hydroxyphenylpyruvate in a homogentisate and which is less sensitive to an HPPD inhibitor agent than the endogenous HPPD of the host plant, such as the HPPD from Arabidopsis thaliana, particularly the HPPD comprising the amino acid sequence of SEQ ID NO. 11 (from amino acid position 126 to amino acid position 568), or comprising the amino acid sequence of SEQ ID NO.11 or SEQ ID NO. 12 (from amino acid position 134 to amino acid position 575).

In a particular embodiment, the present invention relates to a host plant cell, a host plant seed or a host plant, characterized in that it contains at least one nucleic acid sequence encoding an HPPD of the invention as described above. defined in the present context, in which the HPPD of the invention is less sensitive than the endogenous HPPD of the host plant to an herbicide inhibiting the HPPD of the class of isoxazoles, dikettonitriles, tricetonas or pyrazolinates, more especially isoxaflutol, tembotrione, mesotrione, sulcotriona, pirasulfotol, bicyclopyrone, tefuriltrione, topramezone, 2-cyano-3- cyclopropyl-1- (2-SO2CH3-4-CF3phenyl) propane-1,3-dione and 2-cyano-3-cyclopropyl-1 - (2-SO2CH3-4-2,3 Cl2 phenyl) propane-1,3-dione, even more particularly trembling, mesotrione, dicetonitrile, bicyclopirone, topramezone, pyrazolate, benzofenap, sulcotrione, tefuriltrione, and pyrasulfotol, most particularly Larger, mesotrione and bicyclopirone.

In another particular embodiment, the present invention relates to a plant cell or a plant, characterized in that it contains at least one nucleic acid sequence encoding an HPPD of the invention as previously described, and also a chimeric gene comprising a plant-expressible promoter as described above, operatively connected to a nucleic acid sequence encoding a PDH enzyme (prefenate dehydrogenase) (US 2005/0257283).

The present invention also relates to plants containing transformed cells, in particular to plants that are regenerated from transformed cells, and to progeny plants or seeds thereof, which comprise the chimeric HPPD gene of the invention. Regeneration can be obtained by any appropriate procedure, the procedure being dependent on the nature of the species, as described, for example, in the previous references. The following patents and patent applications may be cited, in particular, with respect to the procedures for transforming plant cells and regenerating plants:

US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073,

EP 267,159 A1, EP 604 662 A1, EP 672 752 A1, US 4,945,050, US 5,036,006,

US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346,

US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520,

US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174 A1, EP 486 233 A1,

EP 486 234 A1, EP 539 563 A1, EP 674 725 A1, WO 91/02071 and WO 95/06128.

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The present invention also relates to transgenic plants or parts thereof, which derive by cultivating and / or crossing the above transgenic plants, and to the seeds of transgenic plants, which comprise the chimeric HPPD gene of the invention.

The present disclosure also relates to final products such as flour or oil, which are obtained from plants, parts thereof or from the seeds of the invention.

The transformed plants that can be obtained according to the invention can be of the monocot type, such as wheat, barley, sugar cane, rice, onion, and corn or corn grain, or of the dicot type, such as tobacco, species of soybeans, alfalfa, Brassica spp., plants such as oilseed rape, cotton, sugar beet, clover, vegetables, etc.

The disclosure refers to a process for transforming host organisms, in particular plant or plant cells, by integration into said organisms of at least one nucleic acid sequence or of a chimeric gene as previously defined, in which it is possible obtaining the transformation by any appropriate known means, the means of which are widely described in the specialist literature and, in particular, in the references cited in the present application, for example by use of the vector according to the invention.

A transformation process according to the present invention comprises bombarding cells, protoplasts or tissues with solid or liquid particles to which an aDn has been added, or containing a DNA. Another transformation method comprises using, as the means to transfer to the plant, a chimeric gene that is introduced into an Ti plasmid of Agrobacterium tumefaciens or a Ri plasmid of Agrobacterium rhizogenes. Other methods such as microinjection or electroporation can be used, or otherwise direct gene transfer using PEG. The skilled person can select any appropriate procedure to transform the host organism to be chosen, in particular the plant cell or the plant, such as, for example, the technology for the transformation of soybeans has been extensively described in Examples 1 through 3 disclosed in EP 1186666 A1, incorporated in the present context by reference. For rice, an Agrobacterium-mediated transformation (Hiei et al., 1994 Plant J 6: 271-282, and Hiei et al., 1997 Plant Mol Biol. 35: 205-21, incorporated in the present context by its reference), an electroporation (US 5,641,664 and US 5,679,558, incorporated in the present context by reference), or a bombardment (Christou et al., 1991, Biotechnology 9: 957 incorporated in the present context by its reference). An appropriate technology for the transformation of monocot plants, and particularly rice, is described in WO 92/09696, incorporated in the present context by reference. For cotton, an Agrobacterium-mediated transformation has been described (Gould JH and Magallanes-Cedeno M., 1998 Plant Molecular Biology reporter, 16: 1-10 and Zapata C., 1999, Theoretical Applied Genetics, 98 (2): 1432 -2242 incorporated in this context by reference), transformation mediated by polybrene and / or treatment (Sawahel WA, 2001, - Plant Molecular Biology reporter, 19: 377a-377f).

In a particular embodiment of the invention, the HPPD of the invention is directed towards a target within the chloroplast. This can be done by fusing a nucleic acid sequence encoding a transit peptide with the nucleic acid sequence encoding the HPPD protein of the invention to obtain a nucleic acid encoding a fusion protein as previously described.

Alternatively, the HPPD of the invention can be expressed directly in plastids such as chloroplasts, using a plastidium transformation, such as the chloroplast genome. An appropriate method comprises the bombardment of plant cells or tissues with solid particles coated with the DNA or with liquid particles comprising the DNA, and the integration of the introduced gene encoding the protein of the invention by homologous recombination. Appropriate vectors and selection systems are known to the person skilled in the art. An example of means and procedures that can be used for such integration into the genome of tobacco plant chloroplasts is given in WO 06/108830, the content of which is hereby incorporated by reference.

The present invention also relates to a process for obtaining a plant tolerant to an HPPD inhibitor, characterized in that the plant is transformed with a chimeric HPPD gene of the invention, as previously described.

Therefore, the present invention also relates to a process for obtaining a plant tolerant to an HPPD inhibitor agent, characterized in that the plant contains a chimeric HPPD gene of the invention comprising a coding sequence as well as a heterologous regulatory element. in the 5 'position and optionally in the 3' position, which are capable of functioning in a host organism, characterized in that the coding sequence comprises at least one nucleic acid sequence defining a gene encoding an HPPD of the invention as described above. previously described.

In one embodiment of the present invention, the HPPD inhibitory agent in the above procedure is a tricketone or pyrazolinate type herbicide, preferably trembling, mesotrione, bicyclopyrone, tefuryltrione, pyrasulfotol, pyrazolate, dikettonitrile, benzofenap or sulcotrione, particularly trembling.

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In accordance with the present invention, there is also provided a process for obtaining a plant tolerant to an HPPD inhibitor agent as described above, characterized in that a plant comprising a first transgene is obtained, which is a chimeric gene of HPPD of the invention, and a second transgene, which is a chimeric gene comprising a plant-expressible promoter, operatively connected to a nucleic acid encoding a PDH enzyme (prephenate dehydrogenase). A plant comprising said two transgenes can be obtained by transforming a plant with a transgene, and then re-transforming this transgenic plant with the second transgene, or transforming a plant with the two transgenes simultaneously (in the same DNA or vector or in 2 different DNA or transformation vectors), or crossing a plant comprising the first transgene with a plant comprising the second transgene, as is well known in the art.

The disclosure also refers to a method for selectively removing weeds or preventing germination of weeds in a field where plants are to be planted or in which seeds are sown, or in a crop of plants, by application of a HPPD inhibitor agent to said field or crop of plants, in particular an HPPD inhibitor herbicide as defined above, whose procedure is characterized in that this HPPD inhibitor herbicide is applied to plants that have been transformed according to with the invention, either before sowing the cultivated plant (what is called hereafter application before planting), before the sprout of the cultivated plant (what is hereinafter referred to as application before sprouting), or after outbreak of the cultivated plant (what is called hereafter application after the outbreak).

The present disclosure also relates to a process for growing plants that have been transformed with a chimeric gene according to the invention, which method comprises planting seeds comprising a chimeric gene of the invention, in an area of a field that is appropriate. to cultivate said plants, and apply, if weeds are present, a dose, which is toxic to weeds, of a herbicide whose target is the HPPd defined above to said area of said field, without significantly affecting said transformed seeds or said transformed plants, and then harvest the cultivated plants or parts of plants, when they reach the desired stage of maturity and, when appropriate, separate the seeds from the harvested plants.

In the above procedures, the herbicide whose target is the HPPD enzyme can be applied according to the invention, either before sowing the cultivated plant, before the cultivated plant sprouts or after the cultivated plant sprouts.

The present disclosure also relates to a process for obtaining an oil, particularly a spp soybean oil, corn or cotton, or a flour, comprising growing a cultivated plant, particularly a spp soybean cultivated plant, expressing an HPPD protein of the invention, optionally treating said cultivated plant with an HPPD inhibitor herbicide, harvesting the grains and grinding the grains to produce a flour and extracting the oil. Also the seeds or grains, whether whole, broken or crushed, comprising the chimeric gene of the invention, are part of the present invention.

Therefore, the present invention relates to a process for obtaining an oil or a flour, comprising growing a transformed plant as previously described, optionally treating said plant with an HPPD inhibitor herbicide, harvesting the grains and Grind these grains to produce a flour and extract the oil.

The foregoing methods are further provided in the present invention that involve an HPPD inhibitor herbicide selected from isoxaflutol, trembling, mesotrione, pyrasulfotol, sulcotrione, bicyclopirone, tefuriltrione, topramezone, 2-cyano-3-cyclopropyl-1- (2- Methylsulfonyl-4-trifluoromethylphenyl) -propane-1,3-dione and 2-cyano-1- [4- (methylsulfonyl) -2-trifluoromethylphenyl] -3- (1-methylcyclopropyl) -propane-1,3-dione.

Also provided herein are the foregoing methods of the invention involving an herbicide inhibitor of the HPPD of the tricetonas class, such as trembling, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone , particularly selected from trembling, sulcotrione, topramezone, bicyclopyrone, tefuriltrione and mesotrione, more particularly trembling.

Within the meaning of the present invention, it is understood that a "herbicide" is an active substance such as a herbicide by itself or such a substance that is combined with an additive that alters its effectiveness such as, for example, an agent that increases its activity ( a synergistic agent) or that limits its activity (an antidote). Of course, it should be understood that, for application in practice, the above herbicides are combined, in a manner that is per se known, with the formulation adjuvants commonly used in agricultural chemistry.

HPPD inhibitory herbicides such as those of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected from tremotrione, sulcotrione, topramezone, bicyclopirone, tefuriltrione and mesotrione, more particularly trembling, have an outstanding herbicidal activity against a broad spectrum of economically important monocotyledonous and dicotyledonous annual harmful plants. The active substances also act efficiently on perennial harmful plants that produce stems from rhizomes, rootballs, woody stems or other perennial organs, and that are difficult to control.

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The present disclosure therefore also relates to a method of suppressing unwanted plants or to regulate the growth of plants in plant crops comprising an HPPD according to the invention, in which one or more herbicides inhibitors of HPPDs of the class of tricetonas, such as tremorotrione, sulcotrione and mesotrione, or of the class of pyrazolinates, such as pyrasulfotol and topramezone, particularly selected from tembotrione, sulcotrione, topramezone, bicyclopirone, tefuriltrione and mesotrione, more particularly trembling, is applied ( n) to plants (for example, harmful plants such as weeds or unwanted monocot or dicot cultivated plants), to seeds (for example grains, seeds or vegetative propagules such as tubers or parts of stems with buds) or to the area where plants grow (for example the area under cultivation). In this context, an HPPD inhibitor herbicide of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected from tremotrione, sulcotrione, topramezone, bicyclopirone, Tefuriltrione and mesotrione, more particularly trembling, can be applied for example before planting (if appropriate also by incorporation into the soil), before the outbreak or after the outbreak. Examples of individual representatives of monocotyledonous and dicotyledonous weeds that can be suppressed with an HPPD inhibitor herbicide of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone , particularly selected from trembling, sulcotrione, topramezone, bicyclopyrone, tefuriltrione and mesotrione, more particularly trembling, are mentioned below, without this mention being intended as a limitation to certain species only:

Monocotyledonous harmful plants of the genera: Aegilops, Agropyron, Agrostis, Alopecurus, Apera, Oatmeal, Brachiaria, Bromus, Cenchrus, Commelina, Cynodon, Cyperus, Dactyloctenium, Digitaria, Echinochloa, Eleocharis, Eleusine, Eragrostherara, Eragrostherarachis, Eragrostherarachis , Imperata, Ischaemum, Leptochloa, Lolium, Monochoria, Panicum, Paspalum, Phalaris, Phleum, Poa, Rottboellia, Sagittaria, Scirpus, Setaria, Sorghum.

Dicotyledonous weeds of the genera: Abutilon, Amaranthus, Ambrosia, Anoda, Anthemis, Aphanes, Artemisia, Atriplex, Bellis, Bidens, Capsella, Carduus, Cassia, Centaurea, Chenopodium, Cirsium, Convolvulus, Datura, Desmodium, Emexup, Erysimum, Erysimum, Erysimum Galeopsis, Galinsoga, Galium, Hibiscus, Ipomoea, Kochia, Lamium, Lepidium, Lindernia, Matricaria, Mentha, Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago, Polygonum, Portulaca, Ranunculus, Raphanus, Rorippax, Rotala, Salta, Rotala, Salta Senecio, Sesbania, Sida, Sinapis, Solanum, Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi, Trifolium, Urtica, Veronica, Viola, Xanthium.

In transgenic cultivated plants according to the invention, which comprise an HPPD protein, a DNA or a chimeric gene according to the invention and which can also show one or more additional herbicide resistance against herbicides that differ from herbicide inhibitors of HPPD, the use of an HPPD inhibitor herbicide of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected from tremorione, sulcotrione, topramezone is preferred , bicyclopirone, tefuriltrione and mesotrione, more particularly trembling, in economically important transgenic cultivated plants of useful and ornamental plants, for example cereals such as wheat, barley, rye, oats, sorghum and millet, rice and corn or beet cultivated plants sugar, cotton, soybean species, oilseed rape, potato, tomato, peas and other vegetables.

With regard to plant properties, other than tolerance to HPPD inhibitor herbicides as described in the present invention, conventional routes for the production of new plants, which in comparison with existing plants have properties modified, for example, consist of traditional cultivation and procreation procedures and the generation of mutants. Alternatively, new plants with modified properties can be generated with the aid of recombinant procedures (see, for example, documents EP-A-0221044 A1 and EP-A-0131624 A1). The following have been described, for example, in several cases:

- recombinant modifications of cultivated plants, for the purposes of modifying the starch synthesized in the plants (for example, WO 92/11376, WO 92/14827 and WO 91/19806),

- transgenic cultivated plants, which are resistant to certain herbicides of the glufosinate type (compare, for example documents EP-A-0242236, EP-A-0242246) or the type of glyphosate (document wO 92/00377) or the type of sulfonyl ureas (documents EP-A-0257993 and Us-A-5013659),

- transgenic cultivated plants, for example of corn, cotton or soy species, which are capable of producing toxins of Bacillus thuringiensis (Bt toxins), or hybrids or mutants thereof, which make plants resistant against certain pests (document EP-A-0193259),

- transgenic cultivated plants with a modified fatty acid composition (WO 91/13972),

- genetically modified cultivated plants with new constitutive substances or secondary metabolites, for example new phytoalexins, which establish increased resistance against diseases (EPA documents 309862, EPA 0464461),

- genetically modified plants with reduced photorespiration, which have higher crop yields and a higher tolerance for stress, (EPA 0305398),

- transgenic cultivated plants, which produce important proteins pharmaceutically or diagnostically (in English "molecular pharming" = molecular pharmacology),

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- transgenic cultivated plants, which are distinguished by higher crop yields or better quality,

- transgenic cultivated plants, which are distinguished by a combination of new properties such as a combination of the new properties mentioned above (in English "gene stacking" = gene stacking).

A large number of molecular biology techniques, by means of which new transgenic plants with modified properties can be generated, are known in principle; see for example the quotes of I. Potrykus and G. Spangenberg (editing coordinators) Gene Transfer to Plants, Springer Lab Manual (1995), Springer Berlin, Heidelberg, or Christou's editorial, "Trends in Plant Science" 1 (1996) 423-431).

In order to carry out such recombinant manipulations, it is possible to introduce nucleic acid molecules into plasmids, which allow mutagenesis or modification of the sequences by recombination of DNA sequences. With the help of classical procedures, base substitutions can be carried out, partial sequences can be eliminated or natural or synthetic sequences can be added. For the union of DNA fragments with each other, it is possible to attach adapters or connectors to the fragments; see, for example, quotes from Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; or Winnacker "Gene und Klone", VCH Weinheim 2nd edition of 1996.

The generation of plant cells with a decreased activity of a gene product can be achieved, for example, by the expression of at least one corresponding antisense RNA, an RNA of the same sense to achieve a joint suppression effect, or a combination of RNA. both antisense and in the same sense that it forms a silent double stranded RNA (RNAi) molecule, or by the expression of at least one correspondingly constructed ribozyme, which specifically dissociates transcripts from the aforementioned gene product. To do this, it is first possible to use DNA molecules, which comprise the entire coding sequence of a gene product, including any flanking sequences that may be present, or also DNA molecules, which comprise only parts of the sequence. coding, it being necessary that these parts be long enough to establish an antisense effect on the cells. It is also possible to use DNA sequences that have a high degree of homology with respect to the coding sequences of a gene product, but which are not completely identical.

When nucleic acid molecules are expressed in plants, the protein obtained can be located in any compartment of the plant cell. However, in order to achieve localization in a particular compartment, it is possible, for example, to link the coding region with DNA sequences, which guarantee location in a specific compartment. Such sequences are known to one skilled in the art (see, for example, quotes from Braun et al., EMBO J. 11 (1992), 3219-3227; from Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; and de Sonnewald et al., Plant J. 1 (1991) 95-106). However, nucleic acid molecules can also be expressed in the organelles of plant cells.

Transgenic plant cells can be regenerated according to known techniques to give intact plants. In principle, transgenic plants can be plants of any species of plants, including monocotyledonous or dicotyledonous plants.

Thus, transgenic plants can be obtained which - in addition to the chimeric HPPD gene of the invention - have modified properties as the result of overexpression, suppression or inhibition of homologous (or natural) gene sequences or sequences or an expression of genes or sequences of heterologous genes (= strangers).

In plants, plant cells or seeds of the invention, it is preferred to employ the HPPD inhibitor herbicide of the tricetonas class, such as trembling, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone. , particularly selected from trembling, sulcotrione, topramezone, bicyclopyrone, tefuriltrione and mesotrione, more particularly trembling in transgenic cultivated plants that are also resistant to growth regulating agents such as, for example, dicamba, or against herbicides that inhibit essential plant enzymes, for example acetolactate synthases (ALS), EPSP synthases, glutamine synthases (GS) or hydroxyphenylpyruvate dioxygenases (HPPD), or against herbicides taken from the group consisting of sulfonylureas, glyphosate, glufosinate or benzoylisoxazoles and analogous active substances.

The invention therefore also relates to the use of herbicides applied to these HPPD tolerant plants according to the invention to suppress harmful plants (ie weeds) which also extends to transgenic cultivated plants comprising a second or more resistance or resistance to herbicides in addition to resistance against herbicides inhibitors of HPPD of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, of the isoxazole class such as isoxaflutol or the pyrazolinate class, such as pyrasulfotol and topramezone , particularly selected from trembling, sulcotrione, topramezone, bicyclopyrone, tefuriltrione and mesotrione, more particularly trembling.

The HPPD inhibitory herbicides of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected from treotrione, sulcotrione, topramezone, bicyclopirone, tefuriltrione and mesotrione, more particularly trembling,

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they can be used in the usual formulations in the form of wettable powders, emulsifiable concentrates, atomizable solutions, dusting powders or granules.

The HPPD inhibitory herbicides of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected from treotrione, sulcotrione, topramezone, bicyclopirone, tefuriltrione and mesotrione, more particularly trembling. Examples of possible formulations are wettable powders (WP), water soluble powders (SP), water soluble concentrates, emulsifiable concentrates (EC), emulsions (EW), such as emulsions of the types of oil in water and water in oil, atomizable solutions, suspension concentrates (SC), oil or water based dispersions, oil miscible solutions, encapsulation suspensions (CS), dusting powders (DP), seed disinfectant products, granules for application by spreading and on the ground, granules (GR) in the form of microgranulates, granules formed by atomization, coated granules and granules formed by adsorption, water dispersible granules (WG), water soluble granules (SG), ULV (de ultra-low volume), microcapsules and waxes.

These individual types of formulations are known in principle and are described, for example, in the works of: Winnacker-Küchler, "Chemische Technologie", volume 7, editorial C. Hanser, Munich, 4th edition of 1986; Wade van Valkenburg, "Pesticide Formulations", Marcel Dekker, N.Y., 1973; K. Martens, "Spray Drying Handbook", 3rd edition of 1979, G. Goodwin Ltd, London.

The auxiliary agents required for formulations, such as inert materials, surfactants, solvents and other additive materials, are also known and are described, for example, in the works of: Watkins, "Handbook of Insecticide Dust Diluents and Carriers", 2nd edition , Darland Books, Caldwell NJ; H.v. Olphen "Introduction to Clay Colloid Chemistry", 2nd edition, J. Wiley & Sons, N.Y .; C. Marsden, "Solvents Guide", 2nd edition, Interscience, N.Y. 1963; "Detergents and Emulsifiers Annual" by McCutcheon, MC Publ. Corp., Ridgewood N.J .; Sisley and Wood, "Encyclopedia of Surface Active Agents", Chem. Publ. Co. Inc., N.Y. 1964; Schonfeldt, "Grenzflachenaktive Áthylenoxidaddukte", Wiss. Verlagsgesell., Stuttgart 1976; Winnacker-Küchler, "Chemische Technologie", volume 7, editorial C. Hanser Munich, 4th edition of 1986.

On the basis of these formulations, it is also possible to prepare combinations with other active substances such as pesticides, such as for example insecticides, acaricides, herbicides, fungicides, and with antidotes, fertilizing agents and / or growth regulators, for example in the form of a finished formulation or such as a tank mixture.

Wettable powders are uniformly dispersible formulations in water and which, together with the active substance, also comprise surfactants of ionic and / or non-ionic types (wetting agents, dispersing agents), for example poly (oxyethylated) alkyl phenols, alcohols poly (oxyethylated) fatty acids, poly (oxyethylated) fatty amines, (fatty alcohol) polyglycol ether sulfates, alkane sulphonates, alkyl benzene sulphonates, a sodium lignosulfonate, a 2,2'-dinaphthylmethane-6.6 Sodium disulfonate, a sodium dibutylnaphthalene sulfonate or also a sodium oleoyl methyl taurate, together with a diluent or inert substance. To prepare the wettable powders, the active substances such as herbicides are finely ground, for example in usual equipment such as hammer mills, blow mills and air jet mills and, simultaneously or subsequently, mixed with the auxiliary agents for formulations.

Emulsifiable concentrates are prepared by dissolving the active substance in an organic solvent, for example butanol, cyclohexanone, dimethylformamide, xylene or also higher boiling aromatic compounds or hydrocarbons or mixtures of organic solvents, with the addition of one or various surfactants of ionic and / or non-ionic types (emulsifying agents). Examples of emulsifying agents that can be used are: calcium alkyl aryl sulphonates such as dodecyl benzene sulphonate, or nonionic emulsifiers, such as polyglycol esters with fatty acids, alkyl aryl polyglycol ethers, ( fatty alcohol) polyglycol ethers, condensates of propylene oxide and ethylene oxide, alkyl polyethers, sorbitan esters such as, for example, esters with sorbitan fatty acids, or poly (oxyethylene)-sorbitan esters, such as, for example , poly (oxyethylene) esters with sorbitan fatty acids.

Powders for sprinkling are obtained by milling the active substance with finely divided solid materials, for example, talc, natural clays such as kaolin, bentonite and pyrophyllite, or diatomaceous earth.

Suspension concentrates may be based on water or oils. They can be prepared for example by wet milling by commercially available pearl mills, if appropriate with the addition of surfactants, as already mentioned, for example, above in the cases of the other types of formulations.

Emulsions, for example emulsions of the oil-in-water (EW) type, can be prepared, for example, by agitators, colloid mills and / or static mixers by using aqueous organic solvents and, if appropriate, surfactants, such as them. They have already been mentioned, for example, above for the other types of formulations.

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The granules can be prepared either by projection of the active substance on an inert granulated material, capable of adsorption, or by application of active substance concentrates on the surface of support materials, such as sand, kaolinites, or an inert granulated material with the aid of adhesive agents, for example a polyvinyl alcohol, a poly (sodium acrylate) or also mineral oils. Appropriate active substances can also be granulated in the manner that is usual for the production of granules of fertilizing agents, if desired in admixture with fertilizing agents.

Water dispersible granules are generally prepared by usual procedures, such as spray drying, fluidized bed granulation, tray granulation, mixing with high speed agitators and extrusion without any solid inert material.

For preparing disk granules, fluidized bed granules, extruder granules and spray granules, see, for example, the procedures in "Spray-Drying Handbook" 3rd edition of 1979, G. Goodwin Ltd., London; J.E. Browning, "Agglomeration", Chemical and Engineering 1967, pages 147 et seq; "Perry's Chemical Engineer's Handbook", 5th edition, McGraw-Hill, New York 1973, pages 8-57.

For more details about the formulation of agents for the protection of cultivated plants, see, for example, the works of G.C. Klingman, "Weed Control as a Science," John Wiley and Sons, Inc., New York, 1961, pages 81-96 and J.D. Freyer, S.A. Evans, "Weed Control Handbook", 5th edition, Blackwell Scientific Publications, Oxford, 1968, pages 101-103.

As a general rule, agrochemical formulations comprise from 0.1 to 99% by weight, in particular from 0.1 to 95% by weight, of compounds according to the invention. In wettable powders, the concentration of an active substance is for example, from about 10 to 90% by weight, the remainder up to 100% by weight is made up of the usual constituent formulations. In the case of emulsifiable concentrates, the concentration of an active substance may be about 1 to 90, preferably 5 to 80% by weight. Formulations in the form of dusting powders contain from 1 to 30% by weight of an active substance, preferably in most cases from 5 to 20% by weight of an active substance, and the atomizable solutions comprise about 0, 05 to 80, preferably 2 to 50% by weight of an active substance. In the case of water dispersible granules, the content of an active substance depends in part on whether the active compound is presented in liquid or solid form, and on which are the auxiliary granulation agents, fillers and fillers and the like, which be used. In the case of water dispersible granules, for example, the content of an active substance is between 1 and 95% by weight, preferably between 10 and 80% by weight.

In addition, the aforementioned active substance formulations comprise, if appropriate, auxiliary agents that are conventional in each case, such as adhesive agents, humectants, dispersants, emulsifiers, penetrants, preservatives, frost protection and solvents, fillers and filler, support materials and colorants, antifoams, evaporation inhibitors and pH and viscosity regulating agents.

Based on these formulations, it is also possible to prepare combinations of an HPPD inhibitor herbicide of the tricetonas class, such as trembling, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected of trembling, sulcotrione, topramezone, bicyclopyrone, tefuriltrione and mesotrione, more particularly trembling with other active substances such as pesticides, such as, for example, insecticides, acaricides, herbicides, fungicides, and with antidotes, fertilizing agents and / or growth regulators, for example in the form of a finished formulation or such as a tank mixture, which is to be applied to the plants tolerant to HPPD according to the invention.

The active substances that can be applied to HPPD tolerant plants according to the present invention in combination with an HPPD inhibitor herbicide of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class , such as pyrasulfotol and topramezone, particularly selected from tembotrione, sulcotriona, topramezone, bicyclopirone, tefuriltrione and mesotrione, more particularly trembling in mixed formulations or in a deposit mixture, are, for example, known active substances, which are based on inhibition of, for example, acetolactate synthase, acetyl-CoA carboxylase, cellulose synthase, enolpiruvilshikimato-3-phosphate synthase, glutamine synthetase, p-hydroxyphenylpyruvate dioxygenase, phytoene desaturase, photosystem I, photosystem II, protoporphyrinogen oxidase in Weed Research 26 (1986) 441-445, or in the "The Pesticide Manual", 14th edition, The Briti sh Crop Protection Council and the Royal Soc. of Chemistry, 2003 and the bibliography cited there. Known herbicidal agents or plant growth regulators, which can be combined with the compounds according to the invention, are, for example, the following active substances (the compounds are designated by the common name according to the International Organization for Standardization (ISO) or by chemical name, if appropriate together with the code number) and always comprise all forms of use, such as acids, salts, esters and isomers such as stereoisomers and optical isomers. In this context, one form of use is mentioned by way of example and partly also several forms of use:

acetochlor; acibenzolar, acibenbenzolar-S-methyl, acifluorfen, acifluorphene-sodium, aclonifen, alachlor, alidochlor,

aloxidim, aloxidim-sodium, ametrine, amicarbazone, amidochloro, amidosulfuron, aminocyclopyracloro, aminopyralide,

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60

amitrol, ammonium sulfamate, ancimidol, anilophos, asulam, atrazine, azafenidine, azimsulfuron, aziprotrin, BAH- 043, BAS-140H, BAS-693H, BAS-714H, BAS-762H, BAS-776H, BAS-800H, beflubutamide, benazolin, benazolin-ethyl, bencarbazone, benfluralin, benfuresate, bensulide, bensulfuron-methyl, bentazone, benzofendizona, benzobicyclon, benzofenap, benzofluor, benzoylprop, bifenox, bilanafos, bilanafos-sodium, bispyribac, bispyribac-sodium, bromadlo, bromobutide, bromofenoxim, Bromoxynil, bromuron, buminaphs, busoxinone, butachlor, butafenacil, butamifos, butenachloro, butraline, butroxidim, butylate, cafenstrol, carbetamide, carfentrazone, carfentrazone-ethyl, clomethoxyphene, chloraphene, chloraphene, chloraphene, chloraphrine, chloraphrine, chloraphrine, chloraphene, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphrine, chloraphriflorochlora, chloramphenochlorine, chloramphenochlorine, chloramphenochloride sodium, chlorfenprop, chloroflurenol, chloroflurenol-methyl, chloridazone, chlorimuron, chlorimuron-ethyl, chloromequat-chloride, chloronitrofen, chloroftalim, chlortal-dimethyl, chlorotoluron, chlorosulfuron, cinidon, cinidon-eti It, cinmethyline, cinosulfuron, cletodim, clodinafop, clodinafop-propargyl, clofencet, clomazone, clomeprop, cloprop, clopiralide, chloransulam, chloratram-methyl, cumiluron, cyanamide, cyanazine, cyclanilide, cycloxid, cycloimururon, cycloimururon butyl, cyperquat, ciprazine, ciprazole, 2,4-D, 2,4-DB, daimuron / dimron, dalapon, daminozide, dazomet, n-decanol, desmedifam, desmethrin, detosyl pyrazolate (DTP), di-alato, dicamba , dichlobenyl, dichlorprop, dichlorprop-P, diclofop, diclofop-methyl, diclofop-P-methyl, diclosulam, dietyl, diethyl-ethyl, diphenoxuron, difenzoquat, diflufenican, diflufenzopir, diflufenzopir-sodium, dimefuron, dikeguron dimethylpipethyl , dimethachlor, dimethamethrin, dimetenamide, dimetenamide-P, dimethipine, dimetrasulfuron, dinitramine, dinoseb, dinoterb, diphenamide, dipropetrin, diquat, diquat-dibromide, dithiopyr, diuron, DNOC, eglinazin-ethyl, endothelphthale, etothalphthalate, EPTC, ethanol -meti lo, ethephon, ethidimuron, ethiozine, ethofumesate, ethoxyphene, ethoxyphene-ethyl, ethoxy sulfur, ethobenzanide, F-5331, ie N- [2-chloro-4-fluoro-5- [4- (3-fluoro-propyl) - 4,5-dihydro-5-oxo-1H- tetrazol-1-yl] -phenyl] -ethane-sulfonamide, fenoprop, phenoxaprop, phenoxaprop-P, phenoxaprop-ethyl, phenoxaprop-P-ethyl, fentrazamide, fenuron, flamprop, flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam, fluazifop, fluazifop-P, fluazifop-butyl, fluazifop-P-butyl, fluazolate, flucarbazone, flucarbazone-sodium, flucetosulfuron, fluchloralin, flufenacet (thiafluamide), flufenpyr , flufenpyr-ethyl, flumetralin, flumetsulam, flumiclorac, flumiclorac- pentyl, flumioxazine, flumipropin, fluometuron, fluorodiphene, fluoroglycophene, fluoroglyophene-ethyl, flupoxam, flupropacil, flupropanate, flupirsulfuron, flupirsulfuron, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfurone, flupirsulfuronol ,

flurochloridone, fluroxypyr, fluroxypyr-meptyl, flurprimidol, flurtamone, flutiacet, flutiacet-methyl, flutiamide, phomesaphene, foramsulfuron, forchlorophenuron, phosamin, furyloxyphene, gibberellic acid, glufosinate, glufosinate, glufosinate, glufosinate, glufosinate, glufosinate, glufosinate, liposome , glyphosate-isopropylammonium, H-9201, halosaphene, halosulfuron, halosulfuron-methyl, haloxifop, haloxyfop-P, haloxyfop-ethoxyethyl, haloxyfop-P-ethoxymethyl, haloxyfop-methyl, haloxyfop-P-methyl, hexazinone, HOK-990, HOK-90 -201, HW-02, imazametabenz, imazametabenz-methyl, imazamox, imazapic, imazapir, imazaquina, imazetapir, imazosulfuron, inabenfide, indanofan, indole-acetic acid (IAA), 4-indole-3-yl-butyral (IBA) , iodosulfuron, iodosulfuron-methyl-sodium, ioxynil, isocarbamide, isopropalin, isoproturon, isouron, isoxabeno, isoxaclortol, isoxaflutol, isoxapirifop, KUH-043, KUH-071, karbutylate, ketospiradox, lactofenic acid, lactophene, lactofenic acid, lactophene, lactophene, lactophene, lactophene, lactophene, lactophene, lactofenic acid, lactofenic acid MCPA, MCPB, MCPB-methyl, -ethyl and -sodium, mecoprop, mecoprop-sodium, mecoprop-butotyl, mecoprop-P-butotyl, mecoprop-P-dimethylammonium, mecoprop-P-2- ethyl-hexyl, mecoprop-P-potassium, mefenacet, mefluidide, mepiquat- Chloride, mesosulfuron, mesosulfuron-methyl, metabenzothiazuron, metam, metamifop, metamitron, metazachlor, metazol, methoxypheone, methyldimron, 1-methyl-cyclopropene, methyl isothiocyanate, metobenzuron, metobenzuron, metobromuron, methoxychloromethane, Smethochloromethane metribuzine, metsulfuron, metsulfuron-methyl, molinate, monalide, monocarbamide, monocarbamide dihydrogen sulfate, monolinuron, monosulfuron, monuron, MT 128, MT-5950, that is N- [3- chloro-4- (1-methyl-ethyl) - phenyl] -2-methyl-pentanamide, NGGC-011, naproanilide, napropamide, naptalam, NC-310, that is 4- (2,4-dichloro-benzoyl) -1-methyl-5-benzyloxy-pyrazole, neutron, nicosulfuron , nipiraclofen, nitraline, nitrofen, nitrophenolate sodium (mixture of isomers), nitrofluorphene, nonanoic acid, norflurazone, orbencarb, orthosulfamuron, oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone, oxyfluorfen, paclobutrazol, paraquat dichloride, paraquat, pelargonic acid (nonanoic acid), pendimethalin, pendralina, penoxsulam, pentanocloro, pentoxazone, perfluidone, pethoxamid, phenisopham, phenmedipham, phenmedipham-ethyl, picloram, picolinafeno, pinoxaden, piperofos, pirifenop, pirifenop-butyl, pretilachloro, primisulfuron, primisulfuron-methyl, probenazole, profluazol, prociazine, prodiamine, prifluralin, profoxidim, prohexadione, prohexadione-calcium, promtonrochlorone, pro-pro-prochlorone, calcium, promtonrochlorone, calcium propaquizafop, propazine, propham, propisochlor, propoxycarbazone, propoxycarbazone-sodium, propyzamide, prosulfalin, prosulfocarb, prosulfuron, prynachlor, pyraclonil, pyraflufen, pyraflufen-ethyl, pyrasulfotole, pyrazolynate (pyrazolate), pyrazosulfuron-ethyl, pyrazoxyfen, piribambenz, piribambenz-isopropyl , pyribenzoxime, pyributicarb, pyridafol, pyridate, pyriftalide, pyrimin obac, pyriminobac-methyl, pyrimisulfan, piritiobac, piritiobac-sodium, piroxasulfone, piroxsulam, quinclorac, quinmerac, quinoclamine, quizalofop, quizalofop-ethyl, quizalofop-P, quizalofop-P-ethyl, quizalfurpho-phephelophoplophene, phalxoflufopulphyl, phalxofluoropulphopluphoplophene secbumetone, setoxidim, siduron, simazine, symmetrin, SN-106279, sulcotrione, sulfalate (CDEC), sulfentrazone, sulfometuron, sulfometuron-methyl, sulphosphate (glyphosate-trimesium), sulfosulfuron, SYN-523, SYP-249, SYP-299, SYP-299 SYP-300, tebutam, tebuthiuron, tecnazene, tepraloxydim, terbacil, terbucarb, terbuchlor, terbumeton, terbuthylazine, terbutryn, th-547, thenylchlor, thiafluamide, thiazafluron, thiazopyr, thidiazimin, thidiazuron, thiencarbazone, thiencarbazone-methyl, thifensulfuron, thifensulfuron methyl, thiobencarb, thiocarbazyl, ralkoxidim, trilalate, triasulfuron, triaziflam, triazophenamide, tribenuron, tribenuron-methyl, trichloroacetic acid (TCA), triclopyr, tridiphane, trietazine, trifloxysulfuron, trifloxisulfuron, trifloxysulfuron, trifloxysulfuron, trifloxysulfuron trifluralin, triflusulfuron, triflusulfuron-methyl, trimeturon, trinexapac, trinexapac-ethyl, tritosulfuron, tsitodef, uniconazole, uniconazole-P, vernolate, ZJ-0166, ZJ-0270, ZJ-0543, ZJ-0862 and the following compounds

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image 1

The required application rate of the HPPD inhibitor herbicide of the tricetonas class, such as tremorotrione, sulcotrione and mesotrione, or of the pyrazolinate class, such as pyrasulfotol and topramezone, particularly selected from tremotrione, sulcotrione, topramezone, bicyclopirone , tefuriltrione and mesotrione, more particularly trembling, which is to be applied to areas where plants that are tolerant to HPPD are growing according to the present invention, vary depending on external conditions such as temperature, humidity, nature of the herbicide used and other similar. It may vary, for example, between 0.001 and 1.0 and more kg / ha of active substance, but is preferably between 0.005 and 750 g / ha.

In the case of combined applications of HPPD inhibitor herbicides with herbicides, which differ from HPPD inhibitor herbicides, to HPPD tolerant plants according to the present invention, these mixtures may cause lesions in cultivated plants, based on the presence of non-HPPD inhibitory herbicides. In order to reduce / eliminate such lesions in cultivated plants, appropriate antidotes can be added. These antidotes, which are used in active amounts as antidotes, reduce the side effects of the herbicides / pesticides used, for example in economically important cultivated plants, such as cereals (wheat, barley, rye, corn, rice, millet), Alfalfa, sugar beet, sugar cane, oilseed rape, cotton and soybean species, preferably those of corn, cotton, sugar beet, or soybean species.

The antidotes are preferably selected from the group consisting of:

A) compounds of the formula (S-I)

image2

where the symbols and indexes have the following meanings:

nA is a natural number from 0 to 5, preferably from 0 to 3;

Ra1 is halogen, (C1-C4) alkyl, (C1-C4) alkoxy, nitro or halo (C1-C4) alkyl;

Wa is an unsubstituted or substituted divalent heterocyclic radical, of the group consisting of hetero-

cycles with five-membered rings, partially unsaturated or aromatic with 1 to 3 ring heteroatoms of the No O type, at least one nitrogen atom being present in the ring and at most one oxygen atom, preferably a group radical consisting of in

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(Wa1) to (Wa4),

image3

mA is 0 or 1;

Ra2 is ORa3, SRa3 or NRa3Ra4 or a 3- to 7-membered heterocycle, saturated or unsaturated, which has the

minus one nitrogen atom and up to 3 heteroatoms, preferably of the group consisting of O and S, which is linked through the nitrogen atom with the carbonyl group in (SI), and which is unsubstituted or substituted with radicals of the group that it consists of (C1-C4) alkyl, (C1-C4) alkoxy and optionally substituted phenyl, preferably a radical of formulas ORa3, NHRa3 or N (CH3) 2, in particular of formula ORa3;

Ra3 is hydrogen or an unsubstituted or substituted aliphatic hydrocarbon radical, which preferably has

a total of 1 to 18 carbon atoms;

Ra4 is hydrogen, (Ci-C6) alkyl, (Ci-C6) alkoxy or unsubstituted or substituted phenyl;

Ra5 is H, alkyl (Ci-Cb), halo-alkyl (Ci-Cb), alkoxy (Ci-C4) -alkyl (Ci-Cb), cyano or COORa9, where

Ra9 is hydrogen, (Ci-Cb) alkyl, halo (Ci-Cb) alkyl, (Ci-C4) alkoxy (C1-C4) alkyl, hydroxy (Ci-C6) alkyl, (C3-C12) cycloalkyl or tri-(Ci-C4) alkyl-silyl;

Ra6, Ra7, Ra8 are identical or different, and are hydrogen, (Ci-Cb) alkyl, halo (Ci-Cb) alkyl, (C3-C12) cycloalkyl, or substituted or unsubstituted phenyl;

preferably:

a) dichlorophenylpyrazolin-3-carboxylic acid type compounds, preferably compounds such as ethyl 1- (2,4-dichlorophenyl) -5- (ethoxycarbonyl) -5-methyl-2-pyrazolin-3-carboxylate (Si-i ), ("Mefenpyr-diethyl", see Pestic. Man.) And related compounds, as described in WO 91/07874;

b) dichlorophenylpyrazolcarboxylic acid derivatives, preferably compounds such as ethyl i- (2,4-dichlorophenyl) -5-methylpyrazol-3-carboxylate (S1-2), 1- (2,4-dichlorophenyl) -5-isopropyl- ethyl pyrazol-3-carboxylate (S1-3), 1- (2,4-dichlorophenyl) -5- (1,1-dimethyl-ethyl) pyrazol-3-carboxylate ethyl (s1-4), 1- ( Ethyl 2,4-dichlorophenyl) -5-phenyl-pyrazol-3-carboxylate (S1-5) and related compounds, as described in EP-A-333 131 and EP-A-269 806.

c) compounds of the triazolecarboxylic acid type, preferably compounds such as fenchlorazole (-ethyl ester), that is 1- (2,4-dichlorophenyl) -5-trichloromethyl- (1H) -1,2,4-triazole -3-ethyl carboxylate (S1-6) and related compounds as described in EP-A-174 562 and EP-A-346 620;

d) compounds of the type 5-benzyl- or 5-phenyl-2-isoxazolin-3-carboxylic acids, or 5,5-diphenyl-2-isoxazolin-3-carboxylic acid, preferably compounds such as 5- (2 , Ethyl 4-dichloro-benzyl) -2-isoxazolincarboxylate (S1-7) or ethyl 5-phenyl-2-isoxazolin-3-carboxylate (S1-8) and related compounds, as described herein WO 91/08202, or respectively ethyl 5,5-diphenyl-2-isoxazolin-carboxylate (S1-9) ("isoxadiphene-ethyl") or n-propyl 5,5-diphenyl-2-isoxazolin-carboxylate (S1 -10) or 5- (4-fluoro-phenyl) -5-ethyl phenyl-2-isoxazolin-3-carboxylate (S1-11), as described in patent application WO-A-95 / 07B97 .

B) Quinoline derivatives of the formula (S-II)

image4

where the symbols and indexes have the following meanings:

Rb1 is halogen, (C1-C4) alkyl, (C1-C4) alkoxy, nitro or halo (C1-C4) alkyl; nB is a natural number from 0 to 5, preferably from 0 to 3;

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Rb is ORb, SRb or NRb Rb or a 3- to 7-membered heterocycle,

saturated or unsaturated, which has at least one nitrogen atom and up to 3 heteroatoms, preferably of the group consisting of O and S, which is linked through the nitrogen atom with the carbonyl group in (S-II), and which is unsubstituted or substituted with radicals of the group consisting of (C1-C4) alkyl, (C1-C4) alkoxy or optionally substituted phenyl, preferably a radical of formula ORb3, NHRb4 or N (CH3) 2, in particular of formula ORb3 ;

Rb3 is hydrogen or an unsubstituted or substituted aliphatic hydrocarbyl radical, preferably having a total of 1 to 18 carbon atoms;

Rb4 is hydrogen, (C1-C6) alkyl, (C1-C6) alkoxy or unsubstituted or substituted phenyl;

Tb is an alkanediyl chain (C1 or C2), which is unsubstituted or substituted with one or two radicals (C1-C4) or

with [(C1-C3) alkoxy] -carbonyl;

preferably:

a) compounds of the type 8-quinolinoxyacetic acid (S2), preferably (5-chloro-8-quinolinoxy) -1-methylhexyl acetate (common name "cloquintocet-mexyl" (S2-1), (see Pestic. Man. ),

(5-Chloro-8-quinolinoxy)-1,3-dimethylbut-1-yl acetate) (S2-2),

(5-Chloro-8-quinolinoxy) -4-allyloxybutyl acetate (S2-3),

(5-Chloro-8-quinolinoxy) -1-allyloxypropyl-2-yl acetate (S2-4),

(5-Chloro-8-quinolinoxy) -ethyl acetate (S2-5),

(5-Chloro-8-quinolinoxy) -methyl acetate (S2-6),

(5-Chloro-8-quinolinoxy) -allyl acetate (S2-7),

(5-Chloro-8-quinolinoxy)-2- (2-propylideniminooxy) -1-ethyl (S2-8), (5-chloro-8-quinolinoxy)-2- oxo-propyl acetate (S2-9) acetate ) and related compounds, as described in EP-A-86 750, EP-A-94 349 and EP-A-191 736 or EP-A-0 492 366, and also their hydrates and salts as described in WO-A-2002/034048.

b) compounds of the type of (5-chloro-8-quinolinoxy) malonic acid, preferably compounds such as (5- chloro-8-quinolinoxy) diethyl malonate, (5-chloro-8-quinolinoxy) diallyl malonate, (5 -chloro-8-quinolinoxy) methyl ethyl malonate and related compounds, as described in EP-A-0 582 198.

C) Compounds of formula (S-III)

image5

(S-lll)

where the symbols and indexes have the following meanings:

one

Rc is (C1-C4) alkyl, halo (C1-C4) alkyl, (C2-C4) alkenyl, halo (C2-C4) alkenyl, (C3-C7) cycloalkyl, preferably dichloromethyl;

Rc2, Rc3 are identical or different and are hydrogen, (C1-C4) alkyl, (C2-C4) alkenyl, (C2-C4) alkynyl, halo (C1-C4) alkyl, halo (C2-C4) alkenyl , (C1-C4) alkyl-carbamoyl-(C1-C4) alkyl, (C2-C4) alkenyl-carbamoyl (C1-C4) alkyl, (C1-C4) alkoxy-(C1-C4) alkyl, dioxolanyl-alkyl (C1-C4), thiazolyl, furyl, furyl-alkyl, thienyl, piperidyl, substituted or unsubstituted phenyl, or Rc2 and Rc3 together form a substituted or unsubstituted heterocyclic ring,

preferably an oxazolidine, thiazolidine, piperidine, morpholine, hexahydropyrimidine or benzoxazine ring;

preferably:

Active compounds of the type of dichloroacetamides, which are frequently used as antidotes for pre-outbreak (active antidotes in the soil), such as for example "dichlormide" (see Pestic. Man.) (= N, N-diallyl-2, 2-dichloro-acetamide),

"R-29148" (= Stauffer 3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidone),

"R-28725" (= Stauffer 3-dichloroacetyl-2,2,5-dimethyl-1,3-oxazolidone),

"benoxacor" (see Pestic. Man.) (= 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzoxazine).

"PPG-1292" (= N-allyl-N [(1,3-dioxolan-2-yl) -methyl] dichloroacetamide from PPG Industries),

"DKA-24" (= N-allyl-N - [(allylaminocarbonyl) -methyl] -dichloroacetamide from Sagro-Chem),

"AD-67" or "MON 4660" (= 3-dichloroacetyl-1-oxa-3-aza-spiro [4.5] dean of Nitrokemia or Monsanto respectively),

"Ti-35" (= 1-dichloroacetyl-azepane from TRI-Chemical RT)

"diclonone" (diciclonone) or "BAS145138" or "LAB145138" (= 3-dichloroacetyl-2,5,5-trimethyl-1,3-diazabicyclo [4.3.0] nonano from BASF) and

"furilazole" or "MON 13900" (see Pestic. Man.) (= (RS) -3-dichloroacetyl-5- (2-furyl) -2,2-dimethyl-oxazolidone)

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D) N-Acylsulfonamides of formula (S-IV) and their salts

image6

in which

Xd is CH or N;

Rd1 is CO-NRd5Rd6 or NHCO-Rd7;

Rd2 is halogen, halo (C1-C4) alkyl, halo (C1-C4) alkoxy, nitro, (C1-C4) alkyl, (C1-C4) alkoxy, (C1-C4) alkyl-sulfonyl, (Ci) alkoxy -C4) -carbonyl or (Ci-C4) alkylcarbonyl;

Rd3 is hydrogen, (C1-C4) alkyl, (C2-C4) alkenyl or (C2-C4) alkynyl;

Rd4 is halogen, nitro, (C1-C4) alkyl, halo (C1-C4) alkyl, halo (C1-C4) alkoxy, (C3-C6) cycloalkyl, phenyl, (C1-C4) alkoxy, cyano, alkyl (Ci-C4) -thio, alkyl (Ci-C4) -sulfinyl, alkyl (Ci-C4) -sulfonyl, alkoxy (Ci-C4) -carbonyl or alkyl (Ci-C4) -carbonyl;

Rd5 is hydrogen, (C1-C6) alkyl, (C3-C6) cycloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C5-C6) cycloalkenyl, 3- to 6-membered phenyl or heterocyclyl containing vd heteroatoms of the group consisting of nitrogen, oxygen and sulfur, realizing that the seven radicals mentioned last are substituted by vd substituents, of the group consisting of halogen, (C1-C6) alkoxy, halo (C1-C6) alkoxy, alkyl (Ci-C2) -sulfinyl, alkyl (Ci-C2) -sulfonyl, cycloalkyl (C3-C6), alkoxy (Ci-C4) -carbonyl, alkyl (Ci-C4) -carbonyl and phenyl and, in the case of cyclic radicals are also substituted with (C1-C4) alkyl and halo (C1-C4) alkyl; Rd6 is hydrogen, (C1-C6) alkyl, (C2-C6) alkenyl or (C2-C6) alkynyl, where the three radicals mentioned last are substituted with vd radicals of the group consisting of halogen, hydroxy, (Ci) alkyl - C4), (C1-C4) alkoxy and (Ci-C4) alkylthio, or

RDS and Rd6 form in common with the nitrogen atom carried by a pyrrolidinyl or piperidinyl ring;

Rd7 is hydrogen, alkyl (Ci-C4) -amino, di-alkyl (Ci-C4) -amino, alkyl (C1-C6), cycloalkyl (C3-C6), where the 2 radicals mentioned last are substituted with vd substituents, of the group consisting of halogen, (C1-C4) alkoxy, halogen (C1-C6) alkoxy and (Ci-C4) -thio alkyl and in the case of cyclic radicals, are also substituted with (C1-C4) alkyl ) and halo (C1-C4) alkyl; nD is 0, i or 2; mD is i or 2; vd is 0, i, 2 or 3;

from among these, preference is given to compounds of the N-acyl sulfonamide type, for example of the following formula (S-V), which are known, for example, from WO 97/45016

image7

in which

Rd7 is (C1-C6) alkyl, (C3-C6) cycloalkyl, in which the last 2 radicals are substituted with vd substituents, from the group consisting of halogen, (C1-C4) alkoxy, halogen-C1 (alkoxy) -C6) and alkyl (Ci-C4) -thio and, in the case of cyclic radicals, are also substituted with (C1-C4) alkyl and halo (C1-C4) alkyl;

Rd4 is halogen, (C1-C4) alkyl, (C1-C4) alkoxy, CF3; mD is i or 2; vd is 0, i, 2 or 3;

and also

acylsulfamoylbenzamides, for example of the following formula (S-VI), which are known, for example, from WO 99/16744,

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image8

for example those in which

Rd5 = cyclopropyl and (Rd4) = 2-OMe ("ciprosulfamide", S3-1), Rd5 = cyclopropyl and (Rd4) = 5-Cl-2-OMe (S3-2),

Rd5 = ethyl and (Rd4) = 2-OMe (S3-3),

Rd5 = isopropyl and (Rd4) = 5-Cl-2-OMe (S3-4) and Rd5 = isopropyl and (Rd4) = 2-OMe (S3-5);

and also

compounds of the N-acylsulfamoylphenylureas type of formula (S-VII), which are known, for example, from EP-A-365484

image9

in which

Rd8 and Rd9 independently of each other are hydrogen, (C-i-Cs) alkyl, cycloalkyl (C3-C8), alkenyl (C3-C6), alkynyl (C3-C6),

Rd4 is halogen, (C1-C4) alkyl, (C1-C4) alkoxy, CF3m or is 1 or 2;

of these in particular 1- [4- (N-2-methoxybenzoylsulfamoyl) phenyl] -3-methylurea,

1- [4- (N-2-Methoxybenzoylsulfamoyl) phenyl] -3,3-dimethylurea,

1- [4- (N-4,5-dimethylbenzoylsulfamoyl) phenyl] -3-methylurea,

1- [4- (N-Naphthoylsulfamoyl) phenyl] -3,3-dimethylurea,

G) active compounds of the hydroxyaromatic and aromatic-aliphatic carboxylic acid class, for example ethyl 3,4,5-triacetoxybenzoate, 3,5-dimethoxy-4-hydroxybenzoic acid, 3,5-dihydroxybenzoic acid , 4-hydroxysalicylic acid, 4-fluorosalicylic acid, 1,2-dihydro-2-oxo-6-trifluoromethylpyridin-3-carboxamide, 2-hydroxycinnamic acid, 2,4-dichlorocinnamic acid, as described in WO 2004084631, WO 2005015994, WO 2006007981 and WO 2005016001;

H) active compounds of the 1,2-dihydroquinoxalin-2-one class, for example 1-methyl-3- (2-thienyl) -1,2-dihydroquinoxalin-2-one, 1-methyl-3- ( 2-thienyl) -1,2-dihydroquinoxalin-2-thione, 1- (2-aminoethyl) -3- (2-thienyl) hydrochloride -

1,2-dihydro-quinoxalin-2-one, 1- (2-methylsulfonylaminoethyl) -3- (2-thienyl) -1,2-dihydro-quinoxalin-2-one, as described in WO 2005112630,

I) active compounds that in addition to a herbicidal action against harmful plants, also have an antidote action on cultivated plants such as rice, such as, for example, "dimepiperate" or "MY-93" (see Pestic. Man.) ( = piperidin-1-thio-carboxylate of S-1-methyl-1-phenylethyl), which is known as an antidote for rice against damage caused by the herbicide molinate, "daimuron" or "SK 23" (see Pestic. Man.) (= 1- (1-methyl-1- phenylethyl) -3-p-tolylurea), which is known as an antidote for rice against damage caused by the herbicide imazosulfuron, "cumiluron" = "JC-940" (= 3- ( 2-Chlorophenylmethyl) -1- (1-methyl-1-phenylethyl) urea, see JP-A-60087254), which is known as an antidote for rice against damage caused by some herbicides, "methoxypheone" or "NK 049" (= 3,3'-dimethyl-4-methoxybenzophenone), which is known as an antidote for rice against damage caused by some herbicides, "CSB" (= 1-bromo-4- (chloromethylsulfonyl) benzene) (Reg. CAS 54091 -06-4 from Kumiai), which is known as an antidote against damage caused by some herbicides in rice,

K) compounds of formula (S-IX), as described in WO-A-1998/38856

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in which the symbols and indexes have the following meanings:

Rk1, Rk2 independently of each other, are halogen, (C1-C4) alkyl, (C1-C4) alkoxy, halo (C1-C4) alkyl, (C1-C4) alkyl-amino, di-C1-C4 alkyl ) -amino, nitro;

Ak is COORk3 or COORk4

Rk3, Rk4 independently of each other, are hydrogen, (C1-C4) alkyl, (C2-C6) alkenyl, (C2-C4) alkynyl, cyanoalkyl, halo (C1-C4) alkyl, phenyl, nitrophenyl, benzyl, halobenzyl, pyridinylalkyl or alkylammonium, nK1 is 0 or 1

nK2, nK3 independently of each other, are 0, 1 or 2

preferably: (diphenylmethoxy) methyl acetate (CAS Reg. No .: 41858-19-9),

L) compounds of formula (S-X), as described in WO A-98/27049

image11

in which the symbols and indexes have the following meanings:

Xl is CH or N,

nL in the case that it is X = N, it is an integer from 0 to 4 and in the case that it is X = CH, it is an integer from 0 to 5,

Rl1 is halogen, (C1-C4) alkyl, halo (C1-C4) alkyl, (C1-C4) alkoxy, halo (C1-C4) alkoxy, nitro, (C1-C4) alkyl - thio, (Ci) alkyl -C4) -sulfonyl, (Ci-C4) alkoxycarbonyl, optionally substituted phenyl, optionally substituted phenoxy,

Rl2 is hydrogen or (C1-C4) alkyl,

Rl3 is hydrogen, (Ci-Ce) alkyl, (C2-C4) alkenyl, (C2-C4) alkynyl or aryl, in which each of the above-mentioned carbon-containing radicals is unsubstituted or substituted with one or more , preferably up to three, identical or different radicals taken from the set consisting of halogen and alkoxy; or salts thereof,

M) active compounds of the 3- (5-tetrazolylcarbonyl) -2-quinolones class, for example

1,2-dihydro-4-hydroxy-1-ethyl-3- (5-tetrazolylcarbonyl) -2-quinolone (CAS Reg. No .: 219479-18-2), 1,2-dihydro-4- hydroxy-1-methyl-3- (5-tetrazolyl-carbonyl) -2-quinolone (CAS Reg. No .: 95855-00-8), as described in WO-A-1999000020,

N) Compounds of formulas (S-XI) or (S-XII), as described in WO-A-2007023719 and WO-A- 2007023764

image12

in which

Rn1 is halogen, (C1-C4) alkyl, methoxy, nitro, cyano, CF3, OCF3

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And, Z independently of each other are O or S, nN is an integer from 0 to 4,

Rn2 is (C1-C16) alkyl, (C2-C6) alkenyl, (C3-C6) cycloalkyl, aryl; benzyl, halobenzyl,

Rn3 is hydrogen, (C1-C6) alkyl;

O) one or more compounds of the group consisting of:

1,8-naphthalic anhydride,

O, O-diethyl and S-2-ethylthio-ethyl (disulfoton) phosphorodithioate, 4-chlorophenyl methylcarbamate (methanate), O, O-diethyl-O-phenyl phosphorothioate (dietolate),

4-carboxy-3,4-dihydro-2H-1-benzopyran-4-acetic acid (CL-304415, CAS Reg. No .: 31541-57-8),

1- 2-Propenyl oxa-4-azaspiro [4.5] decane-4-carbodithioate (MG-838, CAS Reg. No .: 133993-74-5), [(3-oxo-1H-2- benzothiopiran-4 (3H) -yliden) methoxy] methyl acetate (from WO-A-98/13361; CAS Reg. No .: 205121-04-6),

cyanomethoxyimino (phenyl) acetonitrile (cyometrinyl),

1,3-dioxolan-2-ylmethoxyimino (phenyl) acetonitrile (oxabetrinyl),

4'-Chloro-2,2,2-trifluoro-acetophenone (fluxophenim) O-1,3-dioxolan-2-ylmethyl-oxime,

4,6-dichloro-2-phenylpyrimidine (fenclorim),

Benzyl chloro-4-trifluoromethyl-1,3-thiazol-5-carboxylate (flurazole),

2-dichloromethyl-2-methyl-1,3-dioxolane (MG-191),

including stereoisomers and the usual salts in agriculture.

A mixture with other known active compounds, such as fungicides, insecticides, acaricides, nematicides, bird repellents, plant nutrients and soil structure enhancing agents, is also possible.

Some of the antidotes are already known as herbicides and, therefore, in addition to the herbicidal action against harmful plants, they also act to protect cultivated plants. The weight ratios of the herbicide (mixture) to the antidote generally depend on the rate of application of the herbicide and the efficacy of the antidote in question and may vary within wide limits, for example in the range of 200: 1 to 1: 200, preferably from 100: 1 to 1: 100, in particular from 20: 1 to 1:20. The antidotes can be formulated in a manner analogous to that of the compounds of formula (I) or their mixtures with other herbicides / pesticides and can be made available and used as a finished formulation or a mixture in deposit with the herbicides.

The required application rate of the compound of the formula (I) varies depending, among other things, on external conditions such as temperature, humidity and the type of herbicide used. It can vary within wide limits, for example between 0.001 and 10,000 or more g / ha of the active substance; however, it is preferably between 0.5 and 5,000 g / ha, particularly preferably between 0.5 and 1,000 g / ha and very particularly preferably between 0.5 and 500 g / ha.

When the transgenic plant of the invention contains one or more different genes for tolerance to other herbicides (such as, for example, a gene encoding a mutated or non-mutated EPSPS that gives the plant tolerance to herbicides of the glyphosate type or a gene of pat or bar that confers tolerance to herbicides of the type of glufosinate), or when the transgenic plant is naturally resistant to another herbicide (such as a tolerance to sulfonylureas), the method according to the invention may comprise simultaneous application or chronologically staggered of an HPPD inhibitor in combination with said herbicide or combination of herbicides, for example herbicides of the types of glyphosate and / or glufosinate and / or sulfonylurea.

The invention also relates to the use of the chimeric gene encoding the HPPD of the invention as a marker gene during the transformation of a plant species, based on the selection of the aforementioned HPPD inhibitor herbicides.

The present invention also relates to a process for obtaining a plant resistant to an HPPD inhibitor agent of the tricetonas or pyrazolinate type, characterized in that the plant is transformed with a chimeric gene that expresses in the plant an HPPD of the invention such as It has been defined in this document.

In a particular embodiment, the invention relates to said process for obtaining a plant resistant to an HPPD inhibitor agent of the tricetonas or pyrazolinate type, characterized in that the HPPD of the invention comprises SEQ ID NO. 4 (from amino acid position 2 to amino acid position 387), or a synthetic DNA encoding the HPPD of the invention adapted to deal with codons of corn, rice, wheat, soybean species, sugar cane, onion, plants of the Brassica species, or cotton.

In another particular embodiment, the invention relates to said process for obtaining a plant resistant to an HPPD inhibitor agent of the type of tricetonas selected from tremotrione, mesotrione, dicetonitrile, isoxaflutol, sulcotrione, tefuriltrione and bicyclopirone.

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In another particular embodiment, the invention relates to said method for obtaining a plant resistant to an HPPD inhibitor agent of the tricetonas or pyrazolinate type, characterized in that the plant also comprises a plant-expressible chimeric gene encoding a PDH enzyme ( prefenate dehydrogenase), or an enzyme with at least PDH.

The disclosure also refers to a process for suppressing weeds in an area or a field, the method of which comprises planting in this zone or field transformed plants resistant to an HPPd inhibitor agent of the type of tricetonas or pyrazolinates, which have been obtained according to the procedure described above, or transformed seeds originating therefrom, and applying a dose, which is toxic to the weeds of said HPPD inhibitor agent of the type of tricetonas or pyrazolinates without significantly affect said transformed seeds or said transformed plants.

The disclosure also refers to a process for obtaining an oil or a flour, comprising growing a transformed plant resistant to an HPPD inhibitor agent of the tricetonas or pyrazolinate type, which has been obtained according to the procedure described above. , or a transformed seed that originates from said plant, optionally treating said plant or seed with an inhibitor agent of HPPd of the pyrazolinate type, harvest the grains and grind the grains to produce a flour and extract the oil.

The invention also relates to the use of an HPPD of the invention as described above, characterized in that the HPPD inhibiting agent is an HPPD inhibitor agent of the tricetonas type, selected from tremothione, mesotrione, topramezone, bicyclopirone, Tefuriltrione and sulcotrione.

The present invention also relates to a host organism, in particular to plant or plant cells, which contain a chimeric gene comprising a sequence encoding an HPPD according to the invention, and also containing a gene that is functional in this host organism allowing overexpression of a prefenate dehydrogenase enzyme (abbreviated herein as PDH).

The term "PDH enzyme," as used herein, refers to any natural or mutated PDH enzyme that exhibits the activity of PDH, converting the prefenate into HPP. In particular, said PDH enzyme can originate from any type of organism. An enzyme with PDH activity can be identified by any procedure that makes it possible either to measure the decrease in the amount of the prefenate substrate, or to measure the accumulation of a product derived from the enzymatic reaction, ie HPP or one of the NADH cofactors or NADPH

Many genes encoding PDH enzymes are described in the literature and their sequences can be identified on the website.
http://www.ncbi.nlm.nih.gov/entrez/. Particularly known is the gene that encodes the PDH enzyme of Saccharomyces cerevisiae yeast (Accession No. S46037) as described in the quotation of Mannhaupt et al. (1989) Gene 85, 303-311, of a bacterium of the genus Bacillus, in particular of the species B. subtilis (Accession No. P20692) as described in the citation of Henner et al. (1986) Gene 49 (1) 147-152, of a bacterium of the genus Escherichia, in particular of the species E. coli (Accession No. KMECTD) as described in the citation of Hudson et al. (1984) J. Mol. Biol. 180 (4), 1023-1051, or of a bacterium of the genus Erwinia, in particular of the species E. herbicola (Accession No. S29934) as described in the citation of Xia et al. (1992) J. Gen. Microbiol. 138 (7), 13091316.

The invention further relates to a process for obtaining a host organism, particularly a plant cell or a plant, which is resistant to an HPPd inhibitor, by integration into said organism of at least one nucleic acid sequence or a gene. chimeric as previously defined, and by subsequent transformation thereof, simultaneously or successively, with a gene that is functional in this host organism allowing the expression of a PDH enzyme (prephenate dehydrogenase). In a particular embodiment, the invention relates to a process for obtaining a host organism, particularly a plant cell or a plant, which is resistant to an HPPD inhibitor agent of the type of tricetonas or pyrazolinates, particularly trembling, mesotrione topramezone, bicyclopyrone, tefuriltrione or sulcotrione.

The means and procedures that could be used to obtain a host organism, particularly a plant cell or a plant, transformed both with a gene that allows overexpression of an HPPD enzyme, and with a gene that allows overexpression of a PDH enzyme, are described extensively in WO 04/024928. The reference made in this specification to any previous publication (or information derived from it) or to any subject that is known, is not taken, nor should it be taken, as an acknowledgment or an admission or any form of suggestion that said previous publication (or previous) or known matter is part of the common general knowledge in the field of the present invention.

Figures

FIGURE 1 Map of plasmid pSE420 :: FMP27e

FIGURE 2 Map of T-DNA introduced into tobacco plants

FIGURE 3 Map of the T-DNA introduced in the different plants according to Examples 5 to 13. Abbreviations having the following meanings A, B, C and G, tobacco plants, D, E, and F, Zea plants mays, H. Soybean plants, I. Rice plants and J, cotton plants. 35S: promoter

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from CaMV35S, KanR: gene that confers resistance to the antibiotic kanamycin, nos: nopaline synthase promoter, Ter: terminator, H6: sequence encoding a His brand, OTP: optimized transit peptide, BAR genes (bialaphic resistant document WO 8705629) and PAT (phosphinothricin N-acetyltransferase, EP 257542): conferring tolerance to bialaphs, phosphinothricin or glufosinate, 2mEPSPS: gene encoding the EPSPS (5-enolpiruvilshikimato synthase) double mutant (Thr102Ile and Pro10a Ser) (US 20030027312), 2mAHAS: gene encoding the double mutant ALS (acetolactate synthase) from Arabidopsis (Pro197Ala and Trp574Leu); US 5378824, HA: histone promoter from the Arabidopsis gene, TEV: tobacco corrosion virus, FMP27e: gene encoding an FMP27 optimized for expression in E coli with a sequence encoding a His tag next to its limb 5 ', FMP27t: gene that encodes an FMP27 optimized for expression in dicotyledonous plants with a sequence that encodes a His tag next to its limb 5', FMP27t-h, a gene that encodes an FMP27 optimized for expression in dicotyledonous plants, FMP27m , a gene that encodes an FMP27 optimized for expression in Zea mays plants, LB, left border, RB, right border.

SEQUENCE LIST

 I KNOW THAT

 ID NO. one:

 I KNOW THAT

 ID NO. 2:

 I KNOW THAT

 ID NO. 3:

 I KNOW THAT

 ID NO. 4:

 I KNOW THAT

 ID NO. 5:

 I KNOW THAT

 ID NO. 6:

 I KNOW THAT

 ID NO. 7:

 I KNOW THAT

 ID NO. 8:

 I KNOW THAT

 ID NO. 9:

 I KNOW THAT

 ID NO. 10:

 I KNOW THAT

 ID NO. eleven:

 I KNOW THAT

 ID NO. 12:

 I KNOW THAT

 ID NO. 13:

 I KNOW THAT

 ID NO. 14:

 I KNOW THAT

 ID NO. fifteen:

 I KNOW THAT

 ID NO. 16:

 I KNOW THAT

 ID NO. 17

 I KNOW THAT

 ID NO. 18

 I KNOW THAT

 ID NO. 19

 I KNOW THAT

 ID NO. twenty

 I KNOW THAT

 ID NO. twenty-one

 I KNOW THAT

 ID NO. 22

 I KNOW THAT

 ID NO. 2. 3

 Examples

Nucleic acid sequence encoding Kordia algicide HPPD

Nucleic acid sequence encoding Kordia algicide HPPD optimized for E. coli, which contains at the 5 'end a nucleic acid encoding an amino acid alanine and 6 amino acids histidine.

Nucleic acid sequence encoding Kordia algicide HPPD optimized for Nicotiana tabaccum, which contains at the 5 'end a nucleic acid sequence encoding an optimized transit peptide and a His tag.

Amino acid sequence of the Kordia algicide HPPD derived from SEQ ID NO. 1 Protein encoded by SEQ ID NO. 2

Amino acid sequence of Kordia algicide HPPD (SEQ ID NO. 4) fused with an OTP (optimized transit peptide (WO 2009/144079))

Protein encoded by SEQ ID NO. 3

Nucleic acid sequence encoding the HPPD of Arabidopsis thaliana Amino acid sequence of the HPPD of Arabidopsis thaliana

Protein encoded by SEQ ID NO. 8 plus an additional alanine directly downstream of the initial amino acid methionine followed by 6 amino acids histidine

Protein of SEQ ID NO. 9 plus the OTP sequence located next to the N-terminal end of the protein

Protein of SEQ ID NO. 10 plus the OTP sequence directly next to the N- end

protein terminal

Xho-OTP-for primer sequence

NcoI-OTP-rev primer sequence

Nucleic acid sequence coding for Kordia algicide HPPD optimized for dicotyledonous plants

 Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

 Zea mays Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

 Brassica napus Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

 Beta vulgaris Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

 Gossypium hirsutum Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

 Glycine max Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

 Hordeum vulgare Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

 Oryza sativa Acid sequence

 nucleic coding for Kordia algicide HPPD optimized for plants

Triticum aestivum

The various aspects of the invention will be better understood with the help of the experimental examples that follow. All the procedures or all the operations described below in these examples are given by way of example and correspond to a choice made between the different procedures that are available to arrive at the same or a similar result. This choice has no effect on the quality of the result and, as a consequence, any appropriate procedure can be used by the skilled person to arrive at the same or a similar result. Most procedures to manipulate DNA fragments are

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described in the work "Current Protocols in Molecular Biology" Volumes 1 and 2, Ausubel F.M. et al., published by Greene Publishing Associates and Wiley Interscience (1989) or in the work Molecular cloning, T. Maniatis, E.F. Fritsch, J. Sambrook, 1982, or in Sambrook J. and Russell D., 2001, Molecular Cloning: a laboratory manual (Third edition)

Example 1

Preparation of the HPPD of Kordia algicida (called FMP27e) of SEQ ID NO. 5 and of the HPPD of Arabidopsis thaliana identified by SEQ ID No. 10.

The coding sequence of the HPPD of Arabidopsis thaliana AtHPPD (1335 bp (base pairs); Genebank AF047834; WO 96/38567) was initially cloned into the expression vector pQE-30 (QIAGEN, Hilden, Germany) between sites of restriction of BamHI and HindIII. The vector obtained was called "pQE30-AtHPPD".

The original Kordia algicide (1164 bp) HPPD sequence encoding the protein listed under the accession number A9DQF2 in UniProtKB / TrEMBL was modified and synthesized using an optimized treatment with Escherichia coli K12 codons (Eurofins MWG operon (Ebersberg, Germany), software (logic program) from GENEius) and was cloned into a modified pBluescript vector (Eurofins MWG operon operon, Ebersberg, Germany). In this vector, the sequence corresponding to the MCS (acronym for multiple cloning site = multiple cloning site) was partially removed so that only the sequences corresponding to the recognition sites by the HindIII restriction enzyme on both sides of the insert remained.

At the 5 'end, directly downstream of the ATG, a nucleic acid sequence encoding an amino acid alanine and a nucleic acid sequence encoding an N-terminal His6 tag (6x HIS, encoded by: cac cat cat cac cat cat) was introduced ). Upstream of the ATG, two additional base pairs of cytosine were added in order to obtain a sequence corresponding to the recognition site of the restriction enzyme NcoI and downstream with respect to the stop codon the sequences corresponding to the recognition site were added of restriction enzyme XbaI. The resulting "pBluescript-FMP27e" vector was digested with the restriction enzymes NcoI and XbaI, the band that did not migrate in the vector size length of approximately 3,000 bp corresponding to the DNA was separated on an agarose gel by electrophoresis. Then, the DNA encoding the HPPD was purified using the MinEluteTM Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned into the pSE420 (RI) NX vector (see below) previously cut with the same restriction enzymes.

The cloning and expression vector pSE420 (RI) NX (5261 bp) is based on the plasmid pSE420 produced by Invitrogen (Karlsruhe, Germany). Modifications of this vector include the addition of an nptII gene (neomycin phosphotransferase; Sambrook and Russell, 2001, Molecular Cloning: a laboratory manual (Third edition)) that confers tolerance to the antibiotic kanamycin and which lacks most of the super - connector region (multiple cloning site).

The plasmid possesses the trp-lac (trc) promoter and the lacIq gene that provides the lac repressor in any E. coli host strain. The lac repressor binds to the lac (lacO) operator and restricts the expression of the target gene; This inhibition can be relieved by induction with isopropyl p-D-1-thiogalactopyranoside (IPTG).

The resulting vector was called "pSE420 (RI) NX-FMP27e" (see Figure 1) and was used to transform Escherichia coli BL21 cells (Merck, Darmstadt, Germany). For the AtHPPD (Arabidopsis thaliana HPPD) that was used as a reference, see WO 2009/144079.

HPPD expression was carried out in E. coli K-12 BL21 containing pQE30-AtHPPD or pSE420 (RI) NX-FMP27e. The cells were allowed to grow until the OD (optical density) reached 0.5, then expression was started from the trp-lac (trc) promoter by induction with 1 mM IPTG that binds to the lac repressor and causes its dissociation from the lac operon. Expression was carried out for 15 h at 28 ° C.

To prepare the pre-starter culture, 2 ml of the TB medium (100 pg * ml'1 carbenicillin) was inoculated with 50 pl of an original E. coli K-12 BL21 load in glycerol. The pre-starter culture was incubated at 37 ° C with stirring at 140 rpm (revolutions per minute) for 15 h. 200 pl of the pre-starter culture were used to start the starter culture (5 ml of TB supplement with 100 pg * l-1), which was incubated for 3 h at 37 ° C.

To prepare the main culture, 400 ml of the TB medium (100 pg * ml-1 carbenicillin) was inoculated with 4 ml of the starter culture. This starter culture was incubated at 37 ° C with shaking at 140 rpm until an OD600 of 0.5 was reached. The expression of the recombinant protein was then induced with 400 μl of a 1 M solution of IPTG. The cells were allowed to grow for an additional hour under these conditions, then the temperature was lowered to 28 ° C and the culture was stirred at 140 rpm for 15 h. The cells were harvested by centrifugation at 6,000 x g for 15 min at 4 ° C. Then the cell pellets were stored at -80 ° C.

Isolation and purification of His6-AtHPPD and His6-FMP27e in native form Cell lysis

The cells were lysed using lysozyme, an enzyme that dissociates 1,4-p bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptideglycan that forms the bacterial cell wall. The cell membranes were then broken by the internal pressure of the bacterial cell. In addition, the lysis buffer contained the Benzonase® Nuclease, which is an endonuclease that hydrolyzes all forms of DNA and RNA without

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damaging proteins and thus greatly reduces the viscosity of the cell lysate. Lysis under natural conditions was carried out on ice.

The QIAexpress® Ni-NTA Fast Start Quick Start Kit was used for the purification of His6-labeled proteins, following the instructions in the user manual.

Purification of His6-labeled proteins by immobilized metal ion affinity chromatography (IMAC)

The cell lysate (10 ml), obtained after centrifugation of the lysis reaction mass, was loaded onto a Ni-NTA Fast Start Column Rapid Initiation Column from the QIAexpress® Ni-NTA Fast Start Kit (Qiagen, Hilden , Germany) and purification was carried out according to the instruction manual. His6-labeled protein was eluted with 2.5 ml of the elution buffer.

Desalination of HPPD solutions by gel filtration

HPPD solutions, eluted from a Ni-NTA Fast Start Fast Start Column with 2.5 ml of an elution buffer, were applied to a Sephadex G-25 PD-10 column (GE Healthcare, Freiburg, Germany ) following the instructions in the user manual. After the entire sample had entered the gel bed, an elution was performed with 3.5 ml of a storage buffer.

The HPPD solutions, eluted from the desalination column, were frozen at -80 ° C in 1 ml aliquots.

Determination of the concentration of the HPPD protein using the Bradford protein assay The protein concentration was determined using the standardized Bradford assay (Bradford, (1976), Anal Biochem 72: 248-254).

Determination of the purity of HPPD solutions using an SDS-PAGE

The integrity of the eluted protein was checked by an SDS-PAGE protein gel electrophoresis using NuPAGE® Novex 4-12% Bis-Tris Gels gel (Invitrogen, Karlsruhe, Germany), approximately 10 pg of protein was loaded. 10 pl of the Laemmli Sample Buffer were added to 1-10 pl of a protein solution and the mixture was incubated at 90 ° C for 10 min. After a short centrifugation operation, the entire mixture was loaded into a slit of an SDS gel previously fixed in an XCell SureLock ™ Novex Mini-Cell gel chamber filled with a NuPAGE® MOPS SDS Running Buffer Elution Buffer (diluted from the 20 x solution with ddH2O). Then a voltage of 150 V was applied to the gel chamber for 1 h. For protein band staining, the gel was immersed in a Coomassie Brilliant Blue R-250 Staining Solution. To fade the poly (acrylamide) gel, it was immersed in a Coomassie Brilliant Blue R-250 Destination Solution until the protein bands appeared blue on a white gel.

Example 2

Kinetic characterization and evaluation of tolerance to HPPD inhibitors of HPDD enzymes “SEQ ID NO. 5 ”and“ SEQ ID NO. 10 ”.

The activity of the HPPD was checked by the classic spectrophotometric test (procedure described extensively in WO 2009/144079).

Determination of in vitro kinetic properties of HPPD

The values of Km, Vmax and kcat for different preparations of HPPD enzymes and the values of Ki, Ki = Kon, and K-1 = Koff for different inhibitors of hPpD were determined using an HPLC assay (liquid phase chromatography of high performance) for measurements of HPPD activity. The test mixtures contained, in a volume of 1 ml, 150 mM of a Tris-HCl buffer at a pH of 7.8, 10 mM of sodium ascorbate, 650 units of bovine catalase (Sigma C30 (Sigma-Aldrich, Munich, Germany), 34 mg of protein / ml, 23,000 units / mg), and appropriate amounts of HPP, purified HPPD enzyme and HPPD inhibitors. For the determination of the values of Km, Vmax and kcat the concentrations of HPP in the test mixture were varied between 10 and 400 pM. For the determination of Ki values, K-i = Kon, and K-1 = Koff, 2 mM of HPP were used. All tests were started by adding an HPPD enzyme to the test mixture and stopped in a series of moments between 0 and 240 s (seconds) by adding 200 pl of the reaction mixture to test tubes for reaction , which contained 20 pl of 10% perchloric acid. The precipitated protein was pelleted by centrifugation for 5 minutes at 10,000 xg. 100 pl of the supernatant material was loaded onto a column of Knauer (Berlin, Germany) Eurospher 100-5 C18 of 250 x 4mm, equilibrated with 10% methanol and 0.1% trifluoroacetic acid (buffer A). The column was eluted, also at a rate of 1.5 ml / min, using a 4 minute wash with buffer A, followed by a 3 minute wash with 95% methanol and a wash for an additional 2 minutes with the buffer A. Elution of HGA (homogentysic acid) and HPP (hydroxyphenylpyruvate) was monitored at 292 nm. The HGA elutes in about 5 minutes and the HPP elutes later. A standard set of HGA concentrations were used to provide a standard curve in order to calibrate the absorbance at 292 nm of the HGA peak as a function of the HGA concentration.

For the determinations of the Km and Vmax values, the initial rates of the HPPD reaction at different substrate concentrations were determined from graphical representations of the HGA formed as a function of the

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time and were coupled with the Michaelis-Menten equation for unreactive enzymes using the succession of ID Business Solutions Ltd. (
www.idbs.com) XLfit software suite. For the determination of the values of Ki, Ki = Kon, and K_1 = Koff the time courses of the HPPD reaction with different concentrations of the inhibitors were coupled with the equations for Mechanism A, competitive inhibition, for tight binding inhibitors. (Cha, S. (1975) Tight-binding inhibitors - I. Kinetic behavior [tight fixation inhibitors - I. Kinetic behavior]. Biochemical Pharmacology 24, 2177-2185) using the succession of logical programs ID Business Solutions Ltd. XLfit software suite room

Table 1

 Kinetic characterization of HPPD enzymes (from Arabidopsis thaliana "SEQ ID NO. 10" and from Kordia algicida "SEQ ID NO. 5") and their respective tolerance to HPPD inhibitors, trembling and dicetonitrile. In Table 1 given below, "Km" (the Michaelis-Menten constant) means the kinetic parameter that is used to characterize an enzyme, and is defined as the concentration of the substrate that allows a semi-maximum reaction rate. Km is further defined as the concentration of the substrate with which the reaction rate reaches half of its maximum value (Vmax / 2) at which Vmax has the meaning of being the maximum reaction rate. Kon = K1 is equal to the binding rate constant of the binding between the enzyme and the substrate and Koff = K-1 is equal to the dissociation rate constant of the enzyme and inhibitor complex. Ki defines the inhibition constant

 HPP Tembotrione Dicetonitrile

 Km Vmax (pM) (pM) (/) -k i * (/> -k i *

 SEQ ID NO. 10

 6.3 1.2 2.3E + 05 3.5E-03 0.015 6.1E + 05 1.1E-02 0.018

 SEQ ID NO. 5

 292 13.6 4.9E + 02 1.6E-02 33 1.1E + 03 1.7E-02 15

In the previous Table 1, it can be seen clearly that while the kinetic parameters Km and Vmax of the HPPD of bacteria “SEQ ID NO. 5 ”and the plant HPPD“ SEQ ID NO. 10 ”already showed significant differences (6.3 pM versus 292 pM) that are even greater in regard to the level of tolerance to tremorotrione (0.015 pM versus 33 pM) and dicetonitrile (0.018 pM versus 15 pM). The HPPD of bacteria “SEQ ID NO. 5 ”was much more tolerant of the HPPD inhibiting agents tested than the plant HPPD“ SEQ ID NO. 10 ”.

Determination of HPPD activity in the presence of various HPPD inhibitors

In this content, the value of pI50 means the logarithm value of the concentration of the inhibitory agent that is necessary to inhibit 50% of the activity of an enzyme in molar concentration.

The pI50 values for the HPPD inhibiting agents were determined from graphical representations of dose and response of the activity of an HPPD as a function of the concentration of the inhibiting agent, using the assay described extensively in WO 2009/144079 in an HPP concentration set at 2 mM and a fixed incubation period of 3 minutes using the succession ID Business Solutions Ltd. XLfit software suite.

Table 2: Determination of the pI50 of HPPD enzymes (from Arabidopsis thaliana "SEQ ID NO. 10" and from Kordia algicide "SEQ ID NO. 5") and their respective tolerances to the various HPPD inhibitors listed below: trembling, dicetonitrile, mesotrione, bicyclopyrone, pyrasulfotol, sulcotrione, pyrazolate, tefuriltrione and benzofenap. The symbol ">>" means that the value was much higher than indicated but could not be calculated exactly within the concentration range of the inhibitor tested (2.5x10'6, 5.0x10'6,

1.0x10-5, 2.5x10-5, 6.3x10-5, 2.5x10'4M).

SEQ ID NO. 10 SEQ ID NO. 5

SEQ ID NO. 10 SEQ ID NO. 5

 Trembling

 Dicetonitrile Mesotrione Bicyclopyrone

 >> 5.6

 >> 5.6

 >> 5.6

 5.2

 5.6

 5.7 >> 5.6 5.0

 Pirasulfotol

 Sulcotriona Pirazolato Tefuriltriona Benzofenap

 5.4

 >> 5.6 5.4 >> 5.6 >> 5.6

 4.9

 > 5.6> 5.6 5.8> 5.6

Table 3: Determination of the percentage of inhibition in the presence of 5.0x10-6 M of inhibitory agents compared to the activity measured in the absence of the inhibitor agent for HPPD originated from Arabidopsis thaliana (SEQ ID NO. 10) and from Kordia Algaecide (SEQ ID NO. 5).

 Tembotrione Dicetonitrile Mesotrione Biciclopirone

 SEQ ID NO. 10

 92 87 86 29

 SEQ ID NO. 5

 81 80 85 38

 Pirasulfotol Sulcotriona Pirazolate Tefuriltriona Benzofenap

 SEQ ID NO. 10

 69 74 61 100 90

 SEQ ID NO. 5

 43 85 89 73 97

In the previous Tables 2 and 3, it can be clearly seen that the bacterial HPPD “SEQ ID NO. 5 ”showed a higher level of tolerance to all HPPD inhibiting agents tested than the plant at all concentrations of HPPD inhibiting agents than was observed using the HPPD“ SEQ ID NO. 10 ”in identical experimental conditions.

Example 3: Construction of chimeric genes for the evaluation of herbicide tolerance of HPPD inhibitors in tobacco plants.

10 A) Construction of the chimeric genes

The vector pRP-RD224 (described extensively in WO 2009/144079) containing the sequence encoding the OTP was used for PCR-mediated binding upstream of the nucleic acid sequence corresponding to the enzyme recognition site XhoI restriction and downstream of the nucleic acid sequence corresponding to the recognition site of the NcoI restriction enzyme. The obtained PCR product was cloned into the pCR®-Blunt II-TOPO® vector (Invitrogen, Karlsruhe, Germany) following the instructions in the user manual. The resulting vector was called "pCR-TOPO-OTP". The insertion of the correct sequence was confirmed by a classical DNA sequencing. The DNA corresponding to the OTP was digested with the restriction enzymes NcoI and XhoI, separated by appropriate gel electrophoresis and previously cloned into plasmid pRT100 (Toepfer, (1987), Nucleic Acid Res 15: 5890) and correspondingly digested with the enzymes 20 of restriction Nco I and XhoI. Plasmid pRT100 contains the CaMV35S promoter and the CaMV35S terminator. The resulting vector was subsequently digested with the restriction enzymes NcoI and XbaI. The vector pSE420 (RI) NX-FMP27e (see Figure 1) was subjected to the restriction enzymes NcoI and XbaI in order to obtain the DNA fragment corresponding to "SEQ ID NO. 2". The resulting vector was digested by use of the HindIII restriction enzyme to subclone the CaMV35S :: OTP :: FMP27e :: CaMV35-term cassette (see Figure 2) 25 into the binary vector pBin19 (Bevan (1984), Nucleic Acid Res 12: 8711-8721.) Previously digested with the same enzyme and dephosphorylated. The resulting vector was called "FMP27ebv".

The pQE-30-AtHPPD vectors were used for the PCR-mediated fixation of an NcoI restriction site and a sequence encoding an N-terminal His6 tag for the 5 'ends and an XbaI restriction site for the 3 ends 'from AtHPPD.

30 The PCR product of the AtHPPD gene was isolated from an agarose gel, cut with the restriction enzymes NcoI and XbaI, purified with the MinElute ™ PCR Purification Kit (Qiagen, Hilden, Germany) and cloned into of the same vector pSE420 (RI) NX cut with the same restriction enzymes.

The generated vector was called "pSE420 (RI) NX-AtHPPD" and was digested with the restriction enzymes NcoI and XbaI and cloned into the previously opened vector pRT100 (Toepfer et al., (1987), Nucleic Acid Res 15: 5890) which contains the CaMV35S promoter and the CaMV35S terminator. The generated vector was called "pRT100-AtHPPD".

The pCR-TOPO-OTP vector was digested with the restriction enzymes NcoI and XhoI, and the DNA band corresponding to the OTP was cloned into the previously opened vector pRT100-AtHPPD with the aforementioned restriction enzymes. The resulting vector was subsequently digested with restriction enzyme 40 HindIII and the expression cassette of interest was cloned into the binary vector pBin19 previously opened and dephosphorylated. The resulting vector was called "AtHPPDbv".

The binary vectors FMP27ebv and AtHPPDbv were used to transform competent Agrobacterium tumefaciens cells (ATHV derived from EHA101) selected on YEB media supplemented with the antibiotics kanamycin and rifampicin (which are described extensively in patent application US005925808A).

45 These Agrobacterium strains containing the binary vectors of interest (FMP27ebv or AtHPPDbv) were used to transform leaf discs from Nicotiana tabacum L. cv Samsun NN tobacco plants, which are approximately 5x5 mm2 in size, as described extensively in the quotation of Horsch et al., (1985), Science 227; 1229-1231.

Leaf discs were grown together for 2 days with Agrobacterium tumefaciens cells containing either the binary vector FMP27ebv or the AtHPPDbv. Then the leaf disks were transferred to a medium that allowed the regeneration of stems for 6 weeks in a medium Ms (from Murashige and Skoog, (1962), Physiol Plant 15 (3): 473-497) supplemented with BAP (1 mg / ml; benzylaminopurine), carbenicillin 5 (250 mg / ml), cefotaxin (250 mg / ml), kanamycin (75 mg / ml) and tremorotrione (10 "6 M)

The regenerated calluses were transferred to a medium in order to induce root development for 6 to 12 weeks: MS (1/2), supplemented with carbenicillin (250 mg / ml), cefotaxin (250 mg / ml), kanamycin ( 75 mg / ml), and tremorotrione (10 "6M). After 6 weeks in this medium, the stems transformed with Agrobacterium tumefaciens cells containing the binary vector AtHPPDbv, were transferred to the same exhausted medium 10 with the inhibitory agent. HPPD trembling.

The results are summarized in Table 5 below.

Throughout the experiment, the plates containing the leaf disks were placed inside a growth chamber under controlled conditions (light for 16 h, night for 8 h, 25 ° C).

Root Callus

The stem calluses regenerated from a cell transformed with a nucleic acid sequence encoding an HPPD comprising SEQ ID NO. 11 (Arabidopsis thaliana) or SEQ ID NO. 7 (Kordia algicida) were transferred to a medium that induced root growth, the medium of which was supplemented additionally with the HPPD trembling agent inhibitor for 6 to 12 weeks. In none of the events containing the HPPD defined by SEQ ID NO. 11 (Arabidopsis thaliana) or did not contain transformed calluses, 20 root growth was observed under the conditions given above. Contrary to this, under identical conditions, calluses containing the HPPD defined by SEQ ID NO. 7 manifestly developed numerous and healthy roots (see Table 4, below).

Table 4

 Calluses containing:

 Selected events for molecular analysis% elongation & rooting in 10 "6 M of tremorione Numbers of events rooted in media without trembling

 SEQ ID NO. eleven

 21 0 5

 SEQ ID NO. 7

 34 62 21

Leaf Disc Regeneration

25 Leaf discs were cut from plants containing HPPDs with SEQ ID NO. 11 (Arabidopsis thaliana) or SEQ ID NO. 7 (Kordia algicida), followed by a regeneration for 6 weeks under classical culture conditions in an MS medium supplemented with BAP (1 mg / ml; benzylaminopurine), carbenicillin (250 mg / ml), cefotaxin (250 mg / ml) and which further comprises one of the HPPD inhibitory agents listed below in the aforementioned concentrations (tremorotrione (10 "° M), dicetonitrile (510-6 M), mesotrione (10-6 M) and bicyclopyrone 30 (10-6 M )), with a medium that does not contain any HPPD inhibitor agent as the positive control.

Experiments the level of regeneration was evaluated as follows:

"-" means that the leaf discs looked the same as a leaf disk from wild-type tobacco plants in a medium supplemented with the aforementioned inhibitors.

"++++" means that leaf discs looked similar to leaf discs from 35 wild-type tobacco plants in a medium without inhibitor.

"+", "++", and "+++" indicate that the regenerated leaf discs were greatly affected (+), moderately (++) and little (+++) by the presence of inhibitors.

The results of the experiments are summarized in Table 5.

Table 5: Effects of various HPPD inhibiting agents on the regeneration of a leaf disc that originates from transgenic plants that comprise a gene encoding an HPPD obtained or from

Arabidopsis (SEQ ID NO. 11) or from Kordia algicida SEQ ID NO. 7.

 Leaf discs containing

 Witness Tembotrione Dicetonitrile Mesotrione Biciclopirone

 SEQ ID NO. eleven

 + + + + - - - -

 SEQ ID No. 7

 + + + + + + + + +

While in the case of plants that contain the HPPD defined by SEQ ID NO. 7 (Kordia algicida) these show an equal or only slightly reduced regeneration compared to this untreated control, the corresponding plants containing the HPPD defined by SEQ ID NO. 11 (Arabidopsis thaliana) do not show any regeneration but develop a clearly visible bleaching phenotype compared to the control

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untreated in the presence of all HPPD inhibitors tested.

Example 4: Greenhouse tests to evaluate the tolerance to HPPD inhibitor herbicides of transgenic tobacco plants expressing a gene encoding a tolerant HPPD protein

Preparation of lineages of transgenic plants expressing HPPD enzymes or Arabidopsis or FMP27. Greenhouse test for herbicide tolerance.

Response to tembotrione, isoxaflutol and bicyclopyrone

T0 tobacco plants that contained either the Arabidopsis gene that encodes an HPPD or the FMP27e gene from Kordia algicida that encodes an FMPD FMP27, mentioned above (Example 3), were transferred to the greenhouse (28/20 ° C ), to develop more and produce seeds. These seeds were harvested and placed on land (ED73 mixed with sand and osmocote Pro) to germinate in the greenhouse (28/20 ° C). Three to four weeks later, some seedlings were transferred to individual pots containing the aforementioned land. Two weeks later, plants with a size of 4-6 cm in diameter were sprayed well with

- trembling at a rate of 100 g of AI / ha prepared from a formulation WP20 (20% wettable powder) supplemented with ammonium sulfate and rapeseed methyl ester, or

- isoxaflutol at a rate of 100 g of AI / ha prepared from a WP20 formulation supplemented with ammonium sulfate and rapeseed oil methyl ester, or

- bicyclopyrone at a rate of 100 g of AI / ha prepared from a WP20 formulation supplemented with ammonium sulfate and rapeseed methyl ester, or

- "blind formulation" prepared from a WP20 formulation without active ingredient (IA) supplemented with ammonium sulfate and rapeseed oil methyl ester, and then transferred to a growth chamber with adequate light conditions (20,000 Lux ).

Seven days after application (DAT acronym for Days After The application) symptoms in transformed plants were evaluated compared to the response observed in wild-type tobacco plants sprayed at the same time and under the same conditions as tobacco plants containing the transgenes (100% means that the plants had the same bleaching phenotype as wild-type plants, 0% means that the plants looked the same as wild-type plants treated with the "blind formulation", and an intermediate percentage represents the degree of the symptoms observed).

Table 6: Wild-type tobacco plants (A) and event populations in tobacco containing alternately, the expression cassettes described above having the CaMV 35S promoter, the sequence encoding an OTP and the sequence that encodes the Arabidopsis HPPD (B), or the CaMV35S promoter, the sequence encoding an OTP, and the FMP27e sequence encoding the HPPD of FMP27 (C). Checks of the damage caused by herbicides at 7 days after application (DAT) by spraying with 100 g of AI / ha of trembling or isoxaflutol supplemented with ammonium sulfate and rapeseed oil methyl ester. It is evident that the plants that contained the FMP27e gene were much more tolerant of trembling and isoxaflutol. Plants belonging to categories (B) and (C) have not been selected in terms of the presence of the respective transgene before the herbicide application.

TO

Wild type

% damage, 7 DAT, 100 g of AI / ha

 Lineage

   Tembotrione Isoxaflutol

 Wt

 1 100 100

 Wt

 2 100 100

 Wt

 3 100 100

 Wt

 4 100 98

 Wt

 5 100 99

 Wt

 6 100 99

 Wt

 7 100 100

 Wt

 8 100 n.d.

 Wt

 9 100 n.d.

 Wt

 10 100 n.d.

 Wt

 11 100 n.d.

 Wt

 12 100 n.d.

 Wt

 13 100 n.d.

 (continuation)

 TO

   % damage, 7 DAT, 100 g of AI / ha

 Wild type Lineage

   Tembotrione Isoxaflutol

 WT |

 14 | 100 n.d

 B

   % damage, 7 DAT, 100 g of AI / ha

 HPPD of Arabidopsis Lineage

   Tembotrione Isoxaflutol

 258

 1 100

 258

 2 100

 258

 3 100

 258

 4 100

 258

 5 100

 258

 6 30

 252

 1 30

 252

 2 40

 252

 3 40

 252

 4 40

 252

 5 50

 252

 6 60

 252

 7 60

 252

 8 70

 252

 9 70

 252

 12 75

 252

 13 75

 252

 14 75

 252

 15 80

 327

 1 10

 327

 2 20

 327

 3 20

 327

 4 40

 327

 5 50

 327

 6 50

 327

 7 70

 327

 8 70

 327

 9 70

 327

 10 70

 327

 11 70

 327

 12 80

 327

 13 80

 327

 14 80

 327

 15 80

100

100

100

100

100

100

30

70

95

98

98

99

99

99

99

100

100

100

100

10

twenty

60

60

70

80

95

98

99

100

100

100

100

100

100

C

FMP27e

% damage, 7 DAT, 100 g of lA / ha

Isoxaflutol Tembotrione Lineage

 121

 1 0 0

 121

 200

 121

 3 0 0

 121

 4 0 0

 121

 5 5 1

 121

 6 5 2

 121

 7 5 20

 121

 8 10 n.d.

 121

 9 10 n.d.

 121

 10 10 n.d.

 121

 11 10 n.d.

 121

 12 10 n.d.

 121

 13 10 n.d.

 129

 1 0 0

 129

 200

 129

 3 5 0

 129

 4 5 0

 129

 5 10 1

 129

 6 10 1

 129

 7 15 1

 129

 8 15 2

 129

 9 30 2

 129

 10 30 2

 129

 11 n.d. 5

 129

 12 n.d. 10

 129

 13 n.d. 10

 218

 1 0 0

 218

 200

 218

 3 0 0

 218

 4 0 0

 218

 5 0 0

 218

 6 0 0

 218

 7 0 0

 218

 8 0 0

 218

 9 0 0

 218

 10 0 1

 218

 11 0 1

 218

 12 5 1

 218

 13 5 2

 218

 14 5 3

 218

 15 5 5

Response to bicyclopirone.

Seeds of wild-type tobacco plants and T1 tobacco plants that were carriers of the FMP27e gene from Kordia algicida encoding an HPPD were sown in an MS medium (Murashige and Skoog 1964) supplemented with 50 g / l kanamycin. After 4 weeks, the rooted green seedlings were transferred 5 to the ground and allowed to grow for 3 weeks in the greenhouse as described above and then sprayed with a mixture containing bicyclopyrone (100 g of AI / ha), sulfate Ammonium and rapeseed oil methyl ester. The plants were classified into two categories based on the phenotype developed in response to the herbicide seven days after treatment. Class I was defined as plants that did not show any damage to light damage in response to herbicide treatment (damage: 0-30%), class II was defined as the 10 plants that presented from heavy damage to damage similar to observed with wild-type plants subjected to the same treatment (damage: 31-100%). In this case only the plants that contained at least one T-DNA were exposed to the herbicide treatment.

In general, it can be seen that plants containing a T-DNA insert showed a significant and sufficient level of tolerance for exposure to a field dose of the bicyclopirone inhibitor HPPD herbicide.

15 Table 7:

Bicyclopirone, 100 g of AI / ha 7 DAT

 Transgene

 Lineage Class I Class II% of tolerant plants

 -

 WT 0 12 0

 FMP27e

 121 90 138 39

 FMP27e

 129 47 111 30

 FMP27e

 218 100 56 64

Plants containing FMP27 HPPD showed tolerance to bicyclopyrone HPPD inhibitor herbicide.

It can be summarized from the tables presented above that the plants that express the FMP27e gene from Kordia algicida that encodes the HPPD of FMP27, obtained from different transgenic events 20 are highly tolerant to several herbicides inhibiting HPPD in doses applied in classical agronomic conditions.

Example 5: Construction of binary vectors to express different optimized variants of dicotyledons in plants and greenhouse testing to assess the tolerance of tobacco plants containing such variants.

25 Cloning in pBin19 of FMP27t (SEQ ID NO. 3), FMP27t-h (SEQ ID NO. 15)

A gene with codon treatment optimized for expression in dicotyledonous plants encoding the HPPD protein FMP27, and called FMP27t-h (SEQ ID NO. 15) and the same gene with an additional sequence encoding an OTP and with a His tag next to its 5 'limb called FMP27t (SEQ ID NO. 3). The sequence corresponding to the FMP27t-h gene was cloned using the restriction enzymes NcoI and XbaI in the 30 pRT100-OTP vector previously described, which contains a promoter and a CaMV35S terminator. The resulting vector was called pRT100-OTP-FMP27t-h. The sequence corresponding to FMP27t was cloned into the previously described pRT100 vector, using the restriction enzymes XhoI and XbaI, and the resulting vector was designated pRT100-OTP-FMP27t. The fragments corresponding to PromCaMV35S-OTP-FMP27t-h-TerCaMV35S and PromCaMV35S- OTP-HIS6-FMP27t-TerCaMV35S were subcloned into the vector pBIN19 (described above) using the restriction enzyme SbfI. The binary vectors were designated respectively pBin19-FMP27t-h (Figure 3C) and pBin19-FMP27t (Figure 3B) and can be used, for example, to transform dicotyledonous plants, such as the tobacco plants described above. Transforming plants that have grown sufficiently are then tested for their tolerance to herbicides that inhibit HPPD, such as trembling. The development of the symptoms observed in response to herbicide treatment is evaluated and compared with the response of wild-type plants under the same conditions.

Transformation of plants, and selection of T0 with 100 g of AI / TBT

As an example, rooted plants that contain PromCaMV35S-OTP-HIS6-FMP27t-TerCaMV35S-T-DNA will be transferred to the greenhouse under classical growth conditions. After an acclimatization time period of two weeks, the T0 plants will be treated with a mixture containing 100 g of tremorothione / ha, prepared from a formulation WP20 (20% wettable powder) supplemented with ammonium sulfate and rapeseed oil methyl ester. Two weeks after treatment, symptoms due to herbicide application will be evaluated. Plants are classified into four categories. Treated plants evaluated as "0" look the same as untreated tobacco plants. The plants evaluated as "1"

they show provisionally bleaching a provisionally light bleaching phenotype due to the application of herbicides. Plants evaluated as "2" have permanent bleaching symptoms from light to strong. Finally, the plants evaluated as "3" have the same appearance as some wild-type tobacco plants subjected to the same treatment. The results are compiled in the following Table 8.

5 Table 8: Response of T0 tobacco plants expressing the FMPD of FMP27.

 Gen

 Number of transformants obtained in a medium containing kanamycin Categories corresponding to the intensity of the symptoms due to the application of tremorotrione at a rate of 100 g of AI / ha on the treated plants

 0 1 2 3

 FMP27t

 42 11 4 10 4

In conclusion, several tobacco plants that express the FMPD of FMP27 are tolerant to trembling.

Example 6: Cloning of the FMP27e, FMP27t and FMP27m genes encoding FMP27 HPPD into a vector to transform Zea mays plants

10 FMP27e (SEQ ID NO. 2), FMP27t (SEQ ID NO. 3), FMP27m-h (SEQ ID NO. 16) a- FMP27e in pHoe6 / Ac: Gene with codon treatment optimized for E. coli, more , next to its 5 'limb, a sequence that encodes an OTP and a sequence that encodes a His tag.

The vector pRT100-FMP27e containing the gene encoding the HPPD of FMP27, optimized for expression in E. coli under the control of the CaMV35S promoter, was digested with the restriction enzyme HindIII.

The CaMV35S :: OTP :: FMP27e :: CaMV35S-term cassette was further cloned into the binary vector pHoe6 / Ac (US 6,316,694) previously digested with the same restriction and dephosphorylated enzyme. The resulting vector was called pHoe6 / Ac / FMP27e.

b- FMP27t in pHoe6 / Ac (SEQ ID NO. 3): Gene with a codon treatment optimized for dicotyledonous plants, plus, next to its 5 'limb, a sequence that encodes an OTP and a sequence that encodes a His tag.

20 FMP27t in pRT100. A version of the gene encoding the FMP27 protein optimized for expression in Nicotiana tobaccum, which also contains at the 5 'end a nucleic acid sequence encoding an optimized transit peptide and a His tag, was commissioned and designated FMP27t. Upstream in relation to this sequence the recognition sequence for restriction enzyme XhoI was added and downstream the recognition sequence for restriction enzyme XbaI was added. The DNAs corresponding to OTP and FMP27t 25 were digested with restriction enzymes XhoI and XbaI, separated by appropriate gel electrophoresis and cloned into the vector pRT100 (Toepfer, (1987), Nucleic Acid Res 15: 5890) previously digested with the XhoI and NcoI restriction enzymes. Plasmid pRT100 contains the CaMV35S promoter and the CaMV35S terminator. The resulting vector was called pRT100-FMP27t, and digested with the restriction enzyme HindIII to separate the DNA corresponding to the CaMV35S :: OTP :: FMP27t :: CaMV35S-term cassette with respect to the rest of the vector, in order to clone it within the previously restricted pHoe6 / Ac vector (US 6,316,694). The resulting vector was called pHoe6 / Ac / FMP27t (Figure 3).

c- FMP27m in pHoe6 / Ac (SEQ ID NO. 18): Gene with a codon treatment optimized for more monocot plants, next to its 5 'limb, a sequence that encodes an OTP. FMP27m in pRT100-OTP (NcoI-XbaI) after HindIII

The gene variant optimized for expression in monocot plants encoding FMP27, called FMP27m, was commissioned, and an NcoI restriction site was added upstream of the initiation codon while the sequence of downstream of the stop codon was added recognition for restriction enzyme XbaI. The DNA sequence corresponding to FMP27m was digested with the restriction enzymes NcoI and XbaI, then separated by gel electrophoresis, and finally isolated from the gel. The isolated DNA fragment was mixed with the pRT100-OTP vector (above) previously digested also with the same restriction enzymes. The resulting vector was designated pRT100-OTP-FMP27m, which contains the CaMV35S :: OTP :: FMP27m :: CaMV35Sterm expression cassette, which was isolated using the HindIII restriction enzyme and then subsequently cloned into the previously open pHOE6 / Ac vector and dephosphorylated, which contains the gene encoding the enzyme PAT (from Phosphinothricin Acetyl Transferase = phosphinothricin acetyl transferase), which confers resistance to the herbicide glufosinate (US 6,316,694). The resulting plasmid was called pHoe / Ac / FMP27m (Figure 3F).

Corn Transformation:

Plasmids pHoe6 / Ac (US 6,316,694), pHoe6 / Ac / FMP27e, pHoe6 / Ac / FMP27t and pHoe6 / Ac / FMP27m were used to transform a corn crop.

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35

40

Four. Five

Corn cultivation, protoplast isolation, transformation and regeneration of fertile transgenic corn plants were carried out in accordance with US Pat. 6284945, "Zea mays (L.) with capability of long term, highly efficient plant regeneration including fertile transgenic maize having a heterologous gene, and their preparation". The transformed calluses were selected on a medium containing phosphinothricin. Then the rooted and regenerated plants were transferred to land and allowed to grow and produce seeds in the greenhouse under classical conditions (28/20 ° C). Adult plants were grown to seed production and the seeds were collected for further planting, and sufficiently developed plants will be treated with the respective HPPD inhibitor herbicides.

Example 7: Construction of a vector containing the FMP27e gene to be expressed in rice plants.

A binary vector for the transformation of rice plants is constructed, for example, with the CaMV35 promoter that drives the expression of the FMP27e gene, with codon treatment optimized for expression in E. coli bacteria and next to its 5 'limb a sequence encoding a His tag was added, and more upstream a sequence encoding an OTP was added followed by the CaMV35S terminator. Additionally, the transformation vector also contains a cassette of the PAT gene in which the gene is driven by a CaMV35S promoter and followed by CaMV35S terminator for a glufosinate based selection during the transformation process (see Figure 3 I) . The binary vector was called pTMV369. A similar binary vector is similarly constructed but comprising an expression cassette that expresses the Arabidopsis gene encoding the HPPD enzyme.

Example 8: Transformation of rice plants.

Rice transformation is achieved using procedures well known in the art. Briefly said, the transformation of rice mediated by Agrobacterium tumefaciens was performed using immature embryos, originating from the 6G4317 restorer lineage. Briefly said, panicles from donor plants were harvested 8-12 days after pollination. The immature seed motto was removed. The seeds were thereafter sterilized using a solution based on NaOCl and Tween. The seeds were previously induced with acetylsalicylic acid. Then Agrobacterium tumefaciens cells are cultured concomitantly with the previously induced seeds in the presence of 4-day acetosyringone at 24 ° C in the dark. After this, the coleoptiles from embryos are removed and washed and then placed on a medium supplemented with phosphinothricin for 3 weeks at 28 ° C with a photoperiod rate of 16 hours. Then the growing calluses were cut from the embryos, and transferred to a fresh medium containing triacillin, phosphinothricin, L-proline and copper (II) sulfate.

For each line of callus and for each concentration of tremor, 3 stems, randomly isolated from different pieces of corns, were transferred to MS / 2 with tremor. As a general rule, the transfer of the stems from the regeneration medium to the MS / 2 took place 9 weeks after the calluses had been placed in the regeneration medium.

The cultures were incubated at 26.5 ° C (16-hour photoperiod) and the evaluation of symptoms was carried out 2 weeks later.

New developing leaves of the transferred stems have been rated on the basis of bleaching and assigned to categories in 3 groups:

a) without bleaching

b) with intermediate bleaching

c) with complete bleaching

Within the category "intermediate bleaching" a distinction has been made between stems that have new leaves, which show only very few symptoms of bleaching and therefore tend to form green leaves, and stems with new leaves almost completely bleached.

Table 9:

 Tremor concentration

   AtHPPD FMP27e

 1 | jM

 No. of stems without bleaching 27 28

 No. of stems with intermediate bleaching

 19 19

 No. of completely bleached stems

 12 10

 5 jM

 No. of stems without bleaching 0 0

 No. of stems with intermediate bleaching

 2 21

 No. of completely bleached stems

 58 36

Response to tembotrione in greenhouse tests.

Rooted T0 seedlings (selected either on phosphinothricin alone or on phosphinothricin supplemented with trembling) were transferred to land in the greenhouse. Following a period of acclimatization time, plants that had grown sufficiently were treated with the different herbicides inhibitors of HPPD. As an example, T0 plants are sprinkled with asbestos of the formulation type WP20 (at a rate of 100 g of AI / ha) supplemented with ammonium sulfate and rapeseed oil methyl ester. Seven days after spray application, symptoms due to herbicide application were evaluated and compared with symptoms observed in wild type plants undergoing the same treatment.

The plants were classified into three categories based on the phenotype developed in response to the herbicide seven days after treatment. Class I was defined as the plants that did not show any damage, class II was defined as the plants that showed slight provisional damage in response to herbicide treatment (damage: 10-40%), class III was defined as plants that presented from heavy damage to damage similar to those observed with wild-type plants subjected to the same treatment (damage: 41-100%).

In general, it can be seen that even plants containing only one T-DNA insert already showed a significant and sufficient level of tolerance at a dose in the exposed field of the herbicide inhibitor of HPPD trembling.

Table 10:

Tembotriona, 100 g of AI / ha 7 DAT

 Transgene

 Number of plants treated Class I Class II Class III

 -

 20 0 0 20

 AtHPPD

 23 1 13 9

 FMP27e

 23 2 17 4

In conclusion, it can be seen that rice plants expressing FMP27 protein are more tolerant to the application of the HPPD inhibitor herbicide trembling than wild-type rice plants.

Example 9: Construction of binary vectors for soybean transformation

A binary vector for the transformation of soybeans is constructed, for example, with the CaMV35 promoter that drives FMP27t-h gene expression (SEQ ID NO. 15), with codon treatment optimized for expression in dicotyledonous plants and together to its 5 'limb, a sequence encoding an OTP was added, and more upstream a sequence of TEV (acronym for Tobacco Etch Virus = tobacco corrosion virus) to improve the stability of mRNA in plants followed by the terminator of CaMV35S. The nucleotide sequence of the FMP27t-h gene is given in SEQ ID NO. 15. Additionally, the transformation vector also contains a cassette of the PAT gene in which the gene is driven by a CaMV35S promoter and followed by a CaMV35S terminator for a glufosinate based selection during the transformation process and a 2mEPSPS gene cassette in which the gene is driven by a histone promoter from Arabidopsis to confer transformed herbicide glyphosate tolerance to transformed plants (see Figure 3 H). The binary vector was called pFCO116.

Example 10: Establishment and selection of T0 soy plants

Soy transformation is achieved using methods well known in the art, such as that described using Agrobacterium tumefaciens mediated transformation of explants of soybean halves, which has been described by Paz et al. (2006, Plant cell Rep. 25: 206). Transformants were identified using isoxaflutol as a selection marker. The appearance of green stems was observed, and documented as an indicator of tolerance to the herbicide isoxaflutol. In total, 1% of the transgenic stems tested showed a normal greening comparable to wild-type soybean stems not treated with isoxaflutol, 40 while wild-type soybean stems treated with the same amount of isoxaflutol were bleached entirely . This indicates that the presence of FMP27 protein makes it possible to tolerate herbicides that inhibit HPPDs, such as isoxaflutol. Tolerant green stems were transferred to rooting or grafted media. Rooted seedlings were transferred to the greenhouse after a period of acclimatization time.

45 Plants containing the transgene were then sprayed with HPPD inhibitor herbicides, such as, for example, with tremorotrione as a rate of 100 g of AI / ha. Ten days after application, the symptoms due to the application of the herbicides were evaluated and compared with the symptoms observed in wild-type plants under the same conditions. Eight events that express the HPPD FMP27 protein have been generated

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twenty

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30

35

40

Four. Five

fifty

55

from the green shoots mentioned above and were transferred to the greenhouse. At four weeks after acclimatization, that is, plants that were in a development stage of 3-4 internodes were treated with 100 g of AI / ha of tremorotrione prepared from a WP 20 formulation supplemented with ammonium sulfate and with the rapeseed oil methyl ester. Ten days after application, the symptoms caused by the application of the HPPD inhibitor herbicide were evaluated and compared with the symptoms observed in treated non-transgenic wild-type soybean plants. Two of the eight events did not show any bleaching phenotype and looked the same as untreated wild-type soybean plants. One event showed slight transient symptoms of bleaching but recovered within 14 days after the application of tremorotrione. The remaining five events exhibited the same bleaching as the non-transgenic wild-type soybean plant after treatment with trembling. All these data confirm that the HPPD FMP27 protein confers tolerance to the HPPD inhibitor herbicides, such as trembling, in soybean plants.

Example 11: Construction of binary vectors for the transformation of cotton.

A binary vector for the transformation of cotton is constructed, for example, with the CaMV35 promoter that drives FMP27t-h gene expression (SEQ ID NO. 15), with codon treatment optimized for expression in dicotyledonous plants and in its 5 'limb was added a sequence encoding an OTP, and further upstream a sequence of TEV (tobacco corrosion virus) was added to improve the mRNA stability in plants, followed by the CaMV35 terminator. The nucleotide sequence of the FMP27t-h gene is given in SEQ ID NO. 15. Additionally, the transformation vector also contains a cassette of the PAT gene in which the gene is driven by a CaMV35S promoter and followed by a CaMV35S terminator for a glufosinate based selection during the transformation process and a cassette of 2mEPSPS gene in which the gene is driven by a histone promoter from Arabidopsis to confer transformed herbicide glyphosate tolerance to transformed plants (see Figure 3 J). The binary vector was called pTSIH25

Example 12: Establishment and selection of T0 cotton plants

The transformation of cotton is achieved using procedures well known in the art, an especially preferred procedure is that described in PCT patent publication WO 00/71733.

Regenerated plants are transferred to the greenhouse. After a period of acclimatization time, plants that have grown sufficiently are sprayed with HPPD inhibitor herbicides such as, for example, trembling at a rate of 100 g of AI / ha, supplemented with ammonium sulfate and rapeseed methyl ester . At seven days after spray application, symptoms due to herbicide treatment are evaluated and compared with symptoms observed in wild-type cotton plants undergoing the same treatment under the same conditions.

Example 13: Construction of binary transformation vectors to generate plants that tolerate four herbicides with different modes of action.

A binary vector for the transformation of dicotyledonous plants is constructed, for example, with the CaMV35 promoter that drives the expression of the FMP27t-h gene (SEQ ID NO. 15), with a codon treatment optimized for expression in dicotyledonous plants and next to its 5 'limb a sequence of an OTP was added, followed by the terminator of CaMV35S. The nucleotide sequence of the FMP27t-h gene is given in SEQ ID NO. 15. Additionally, the transformation vector also contains a PAT gene cassette in which the gene is driven by a CaVM35S promoter and followed by a CaMV35S terminator to confer to the plant expressing the gene a tolerance to glufosinate, a 2mEPSPS gene cassette encoding the double mutant EPSPS (Thr102Ile and Pro106Ser) in which the gene is driven by a histone promoter from Arabidopsis to confer transformed herbicide glufosinate tolerance to the transformed plants, and a 2mAHAS gene cassette Arabidopsis thaliana encoding a tolerant ALS enzyme (acetolactate synthase, Pro197Ala, Trp574Leu) driven by a CaMV35S promoter in order to confer to the plant expressing this gene a herbicide tolerance of the sulfonylureas and imidazolinones classes (see Figure 3 G).

The gene cassettes are finally cloned into the pHoe6 / Ac vector (US 6,316,694), and the final vector is called pHoe6 / FMP27t-h / PAT / EPSPS / AHAS, and is used to transform dicotyledonous plants from procedures of the prior art mediated by Agrobacterium tumefaciens. T0 plants are transferred to land, and after a period of acclimatization time, plants that have grown sufficiently are sprayed successively with a herbicide of the class of HPPD inhibiting agents, then with glyphosate, then with glyphosate and finally with a herbicide of the sulphonylureas class, for example.

Example 14: Generation of transgenic plants that show tolerance to herbicides with three different modalities of action.

A binary vector for tobacco transformation is constructed, for example, with the CaMV35 promoter that drives FMP27t-h gene expression (SEQ ID NO. 15), with codon treatment optimized for expression in dicotyledonous plants and together to its 5 'limb a sequence encoding an OTP was added, and further upstream a sequence of TEV (tobacco corrosion virus) was added to improve the mRNA stability in plants, followed by the CaMV35 terminator. The nucleotide sequence of the FMP27t-h gene is given in SEQ ID

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30

NO. 15. Additionally, the transformation vector also contains a cassette of the PAT gene in which the gene is driven by a CaMV35S promoter and followed by a CaMV35S terminator for a glufosinate based selection during the transformation process and a cassette of 2mEPSPS gene in which the gene is driven by a histone promoter from Arabidopsis to confer transformed herbicide glyphosate tolerance to transformed plants (see Figure 3 H). The binary vector was called pFCO116. The above vector was used to transform leaf discs obtained from Nicotiana tobacum plants, according to Example 3.

Transgenic tobacco plants were transferred to the greenhouse and treated with glyphosate at a rate of 1,121 g of AI / ha. Seeds were produced from these tolerant tobacco plants and harvested. These seeds will be placed on land to germinate in the greenhouse. Three to four weeks later, 50 seedlings per event are transferred to individual pots. Two weeks later, plants with a size of 4-6 cm are sprayed, respectively, with:

- glufosinate-ammonium 1,000 g of AI / ha

- glyphosate 1,121 g of AI / ha

- shake 100 g of AI / ha, or

- tembotriona + glyphosate 100 g of AI / ha + 1,121 g of AI / ha.

After nine days, the symptoms caused by the respective herbicide applications were evaluated, as compiled in Table 11, below.

The plants were classified into three categories based on the phenotype developed in response to the respective herbicide or herbicides seven days after treatment. Class I was defined as plants that did not show any damage. Class II was defined as the plants that presented slight provisional damages in response to herbicide treatment (damage: 10-40%). Class III was defined as plants that presented from heavy damage to damage similar to those observed with wild type plants subjected to the same treatment (damage: 41-100%). Non-transgenic tobacco plants were completely annihilated by a treatment using one or more of the above herbicides.

Table 11:

 Transgene

 Lineage Glufosinate ammonium 1,000 g of AI / ha 9 DAT Class I Class II Class III

 FMP27t-h

 639 10 0 0

 FMP27t-h

 714 10 0 0

 FMP27t-h

 717 10 0 0

 Transgene

 Lineage Class I Glyphosate 1,121 g of AI / ha 9 DAT Class II Class III

 FMP27t-h

 639 9 0 1

 FMP27t-h

 714 10 0 0

 FMP27t-h

 717 10 0 0

 Transgene

 Lineage Class I Tembotriona 100 g of AI / ha 9 DAT Class II Class III

 FMP27t-h

 639 7 3 0

 FMP27t-h

 714 5 1 4

 FMP27t-h

 717 4 2 4

Glyphosate + trembling 1,121 g of AI / ha + 100 g of AI / ha 9 DAT

 Transgene

 Lineage Class I Class II Class III

 FMP27t-h

 639 8 2 0

 FMP27t-h

 714 4 3 3

 FMP27t-h

 717 1 1 8

All these data confirm that these plants encoding various tolerance genes that concern herbicides that inhibit HPPD, glyphosate and glufosinate-ammonium, confer tolerance to any of these herbicides applied alone or in combination with each other.

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This is the first time that transgenic plants that contain these three tolerance genes in a single place (locus) exhibit a herbicide tolerance to such herbicides at an application rate that is of agronomic relevance.

SEQUENCE LIST

<110> Bayer CropScience AG Bayer CropScience AG

<120> HPPD-inhibiting herbicide-tolerant plants <130> BCS 09-1043-PCT <160> 23

<170> PatentIn version 3.3

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 atggcagcag

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 aaaattcgct

 tggtcttaac aacaccatta ggaaaaggtg gcgaaatcaa tgagcatatc 300

 gatttacacg

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 tttgaagaaa

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 aatggaaatg

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 caaattgaag

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 catttggagt aggaaacttt aaagctttat ttgaagcgat agaaagagaa 1140

 caagctgctc

 gcggaacatt gtaa 1164

<210> 2 <211> 1185 <212> DNA

5

10

fifteen

twenty

25

<213> Artificial sequence <220>

<223> Nucleic acid sequence encoding HPPD of Kordia algicide optimized for E. coli, which also contains at the 5 'end a nucleic acid encoding an alanine and 6 histidine amino acids.

<220>

<221> misc_feature <222> (4) .. (6)

<223> sequence encoding Ala <220>

<221> misc_feature <222> (7) .. (24)

<223> sequence encoding a His tag containing 6 His <400> 2

 atggctcatc

 atcatcacca tcatgcggcc gaaatcaaga acctgaaaga tttgcagaat 60

 accgaatacg

 gccttaagaa actgtttgac gaggctgagg acttcttgcc gttactgggt 120

 acggattatg

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 gcgtttgggt

 tccaatccga agcgtatgca ggtctggaga caggcctgac cgatcgtgtg 240

 agttacgtgc

 tgaaacagga taaaatccgc ttagtcttaa ctactccact gggtaaagga 300

 ggtgagatta

 acgaacacat tgaccttcat ggtgatggtg tgaaagtggt tgccttatgg 360

 gtcgaagatg

 cgacaaaagc gtttgaggag actaccaaac gcggagccaa accgtatatg 420

 gaacccacca

 aagaagagga cgaaaacggc tatgtcattc gcagcggcat ctatacgtat 480

 ggcgaaaccg

 ttcacgtgtt tgtagaacgc aagaactaca acggagtttt cttgcctggg 540

 taccaacgtt

 gggaaagtca ctacaatcca gaaccggtag gcctcaaatt cattgaccat 600

 atggttggca

 atgttgggtg gggtgaaatg aaagaatggt gcgaattcta tgcgaaagtg 660

 atgggtttcg

 cacagatcat ctcgtttacc gacgacgata tttcgaccga tttcactgct 720

 ctgatgagca

 aagttatgtc caatggcaat ggccgcatta agttcccgat taacgaacct 780

 gccgaaggca

 agaagaaaag ccagattgag gagtatcttg acttttacaa tggctctggt 840

 gtgcaacaca

 ttgcagttgc caccgataac atcatcgata ccgtgagcca gatgcgcgaa 900

 cgtggtgtcg

 agtttctgta tgtaccggac acgtattacg atgatctgct ggaacgggta 960

 ggcgatatcg

 acgaagatgt cgaagaactg aaaaaacatg ggatactcat tgatcgggat 1020

 gaagagggct

 atttgctcca actgtttacc aaaacgatcg tggatcgtcc gacgatgttc 1080

 tttgaagtga

 tacagcgcaa aggagcacag tcatttggcg tggggaactt taaagcgctg 1140

 tttgaagcga

 ttgagcgcga acaagcagcg cgtggtacac tgtaa 1185

<210> 3 <211> 1560 <212> DNA

<213> Artificial sequence <220>

<223> Nucleic acid sequence encoding HPPD of Kordia algicide optimized for Nicotiana tobaccum which also contains at the 5 'end a nucleic acid encoding an optimized transit peptide and a His tag.

10

fifteen

twenty

<220>

<221> TRANSIT_peptide <222> (1) .. (375)

<223> Optimized transit peptide for chloroplasts <220>

<221> misc_feature <222> (376) .. (378)

<223> sequence encoding a Met <220>

<221> misc_feature <222> (379) .. (381)

<223> sequence encoding a <220> Wing

<221> misc_feature <222> (382) .. (399)

<223> sequence encoding a His tag containing 6 His <400> 3

 atggcttcta

 tttcttcttc tgtggctact gtttctagga ctgctccagc tcaagctaat 60

 atggtggctc

 cattcacagg cttgaaatcc aatgctgctt tcccaactac taagaaggct 120

 aacgatttct

 ctactctccc atctaatggt ggaagggttc agtgtatgca agtttggcca 180

 gcttacggaa

 ataagaagtt cgagactctt tcttaccttc caccactttc tatggctcca 240

 actgtgatga

 tggcttcttc tgctactgct gttgctccat tccaaggatt gaagtctact 300

 gcttctttgc

 cagttgctag aaggtcatct cgttctcttg gaaacgtttc taacggtgga 360

 aggattagat

 gtgctatggc tcatcatcat caccatcacg ctgctgagat taagaacctc 420

 aaggatctcc

 agaatactga gtacggactc aagaaacttt ttgatgaggc tgaggatttc 480

 cttccacttc

 tcggaactga ttacgttgag ctttatgtgg gaaacgcaaa gcaatctgct 540

 cacttctaca

 agactgcttt cggatttcaa tctgaggctt acgctggact tgaaactgga 600

 cttactgata

 gggtttccta cgtgcttaag caggataaga ttaggcttgt gctcactact 660

 ccacttggaa

 agggtggaga gattaacgag cacattgatc ttcatggtga tggtgttaag 720

 gttgtggctc

 tttgggttga agatgctact aaggctttcg aagagactac taagagaggt 780

 gcaaagcctt

 atatggaacc tacaaaagaa gaggacgaga acggatacgt gattagatcc 840

 ggaatctaca

 cttacggtga aactgttcac gttttcgtgg agaggaagaa ctacaacgga 900

 gtctttcttc

 ctggatacca acgatgggag tctcattaca atccagagcc agtgggactt 960

 aagttcatcg

 atcacatggt gggtaatgtt ggatggggag agatgaagga atggtgcgag 1020

 ttttacgcta

 aggttatggg attcgctcag atcatttcct tcactgatga tgatatctcc 1080

 actgatttca

 ctgctcttat gtccaaggtg atgtctaatg gaaacggaag gatcaagttc 1140

 cctattaacg

 aaccagctga gggaaagaag aagtcccaga tcgaagagta cctcgatttc 1200

5

 tacaacggat

 ctggtgttca gcatattgct gtggcaactg ataacatcat cgatactgtg 1260

 tctcaaatga

 gagaaagggg agtggagttt ctttacgtcc cagatactta ctacgatgat 1320

 ctccttgaga

 gagtgggaga tattgacgag gatgtggagg aacttaagaa gcacggaatc 1380

 ctcattgata

 gagatgaaga gggatacctt ctccagcttt tcactaagac tatcgtggat 1440

 aggccaacta

 tgttcttcga agtgatccaa agaaagggtg ctcaatcttt cggagtggga 1500

 aacttcaagg

 ctcttttcga ggctattgag agagaacaag ctgctagagg aactctttga 1560

<210> 4 <211> 387 <212> PRT <213> Kordia algaecide

<400> 4

image13

image14

<210> 5 <211> 394 5 <212> PRT

<213> Artificial sequence

<220>

<223> encoded by SEQ ID NO. 2

10

<220>

<221> MISC_FEATURE <222> (2) .. (2)

<223> Wing

<220>

<221> MISC_FEATURE <222> (3) .. (8)

5 <223> His tag made of 6 His

<400> 5

image15

5

10

Gln lie lie Ser Phe Thr Asp Asp 225 230

Leu Met Ser Lys Val Met Ser Asn 245

Lie Asn Glu Pro Wing Glu Gly Lys 260

Leu Asp Phe Tyr Asn Gly Ser Gly 275 280

Asp Asn lie lie Asp Thr Val Ser 290 295

Phe Leu Tyr Val Pro Asp Thr Tyr 305 310

Gly Asp lie Asp Glu Asp Val Glu 325

lie Asp Arg Asp Glu Glu Gly Tyr 340

Lie Val Asp Arg Pro Thr Met Phe 355 360

Gln Wing Ser Phe Gly Val Gly Asn 370 375

Glu Arg Glu Gln Wing Wing Arg Gly 385 390

Asp lie Ser Thr Asp Phe Thr Ala 235 240

Gly Asn Gly Arg lie Lys Phe Pro 250 255

Lys Lys Ser Gln lie Glu Glu Tyr 265 270

Val Gln His lie Ala Val Ala Thr 285

Gln Met Arg Glu Arg Gly Val Glu 300

Tyr Asp Asp Leu Leu Glu Arg Val 315 320

Glu Leu Lys Lys His Gly lie Leu 330 335

Leu Leu Gln Leu Phe Thr Lys Thr 345 350

Phe Glu Val lie Gln Arg Lys Gly 365

Phe Lys Ala Leu Phe Glu Ala lie 380

Thr leu

<210> 6 <211> 512 <212> PRT

<213> Artificial sequence <220>

<223> Kordia algicide HPPD amino acid sequence (SEQ ID NO. 4) fused to an optimized transit peptide (WO 2009/144079)

<220>

<221> TRANSIT <222> (1) .. (125)

<223> Optimized transit peptide for chloroplasts <400> 6

Met Ala Ser lie Ser Ser Ser Val Val Thr Val Ser Arg Thr Ala Pro 15 10 15

Claims (10)

5 10 fifteen twenty 25 30 35 1. A chimeric gene comprising a coding sequence operably linked to a plant-expressible promoter, the latter being a heterologous regulatory element to the coding sequence, characterized in that the coding sequence comprises a nucleic acid sequence that encodes an HPPD protein comprising the amino acid sequence of SEQ ID NO. 4 from amino acid position 2 to amino acid position 387 or a protein with a sequence identity of at least 80% with the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 387, which shows the properties of catalyzing the conversion of para-hydroxyphenylpyruvate to homogentisate and being less sensitive to an HPPD inhibitor herbicide than the endogenous HPPD of the host plant. 2. The chimeric gene according to claim 1 characterized in that it comprises a nucleic acid sequence that encodes an active transit peptide in plants such that a fusion protein of the transit peptide / HPPD is encoded by said chimeric gene. 3. A vector comprising at least one chimeric gene according to any one of claims 1 to 2. 4. A plant cell, part of a plant, plant or seed, characterized in that it comprises a chimeric gene according to any one of claims 1 to 3. 5. The plant cell, part of the plant, plant or seed of claim 4, which also comprises a chimeric gene encoding a PDH enzyme (prefenate dehydrogenase). 6. The plant cell, plant, plant or seed part of claim 4 or 5 further comprising one or more chimeric genes that confer tolerance to growth regulators, preferably 2,4-D or dicamba and / or herbicides that inhibit acetolactate synthase (ALS), EPSP synthase (EPSPS) and / or glutamine synthase (GS). 7. A method of obtaining a plant tolerant to an HPPD inhibitor herbicide, characterized in that a chimeric gene is introduced into said plant according to any one of claims 1 to 2. 8. A method of obtaining an oil or a flour comprising growing a plant according to claim 4, 5 or 6, optionally treating said plant with an HPPD inhibitor herbicide, harvesting the grains and grinding the grains to produce the flour and optionally extract the oil. 9. Use of an HPPD protein comprising the amino acid sequence of SEQ ID NO: 4 or an HPPD protein with a sequence identity of at least 80% to the sequence of SEQ ID NO: 4 and whose HPPD protein shows the properties of catalyzing the conversion of para-hydroxyphenylpyruvate to homogentisate to make the plants tolerant to herbicides that inhibit HPPD. 10. The use according to claim 9, wherein the HPPD inhibitory herbicides are selected from the group consisting of: isoxaflutol, trembling, mesotrione, sulcotrione, pyrasulfotol, topramezone, 2-cyano-3- cyclopropyl-1- (2-SO2CH3-4-CF3phenyl) propane-1,3-dione and 2-cyano-3-cyclopropyl-1- (2-SO2CH3-4-2,3 Cl2 phenyl) propane-1,3-dione, bicyclopyrone, benzobiciclone, tefuriltrione, dicetonitrile and pyrazoxyphene. rbs2 image 1 Figure 1: plasmid map pSE420-FMP27e FMP27c CaMV35S terminator! . CaMV35S screwdriver H useful II OTP Hxllll image2 T-DNA :: FMP27e Figure 2: T-DNA map inserted in tobacco plants image3 image4

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II ^ or LU image5 image6 Figure 3: map of the different DNA stained in image7